WO2018011739A1 - Virus particle linked to carbohydrate antigen for differential diagnosis of chagas disease, method, kit, vaccine and use - Google Patents

Virus particle linked to carbohydrate antigen for differential diagnosis of chagas disease, method, kit, vaccine and use Download PDF

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WO2018011739A1
WO2018011739A1 PCT/IB2017/054219 IB2017054219W WO2018011739A1 WO 2018011739 A1 WO2018011739 A1 WO 2018011739A1 IB 2017054219 W IB2017054219 W IB 2017054219W WO 2018011739 A1 WO2018011739 A1 WO 2018011739A1
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gal
chagas disease
diagnosis
protein
alpha
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Portuguese (pt)
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Alexandre FERREIRA MARQUES
Ricardo Tostes Gazzinelli
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Universidade Federal De Minas Gerais - Ufmg
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/005—Trypanosoma antigens
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is directed to a Qf-VLP bacteriophage coupled to several copies of the synthetic alpha-Gal trisaccharide (Gal 1 -3Gal ⁇ f -1 -4GlcNAcR), whether or not associated with recombinant proteins such as Q ⁇ -Gal. AdASP-2, and its use as a vaccine particle.
  • the present invention also describes a method and kit for detection of anti-alpha-Gal antibodies by ELISA for diagnosis of Chagas disease.
  • the method of the present invention enhances the capture of antibodies produced by exposure to T. cruzi, exhibiting high sensitivity and selectivity and enabling detection of infection and monitoring the effectiveness of benznidazole treatment.
  • Chagas disease or American Trypanosomiasis, is a disease caused by the protozoan Trypanosoma cruzi. According to the World Health Organization, an estimated 7 million people are infected worldwide (WHO. 2015. Chagas disease (American trypanosomiasis), on World Health Organization. Http: // www.who.int/ mediacentre / factsheets / fs340 / en /), with approximately 5.7 million cases in Latin America and approximately 30,000 new cases per year (WHO. 2015. Chagas disease in Latin America: an epidemiological update based on 2010 estimates. Wkly Epidemiol Rec 90: 33-43).
  • the disease represents a major public health problem and an economic problem, as most patients in the chronic stage of the disease are at the most economically active age, in addition to the high costs of treatment and vector control strategies (Moncayo A, Silveira AC. 2009. Current epidemiological trends for Chagas disease in Latin America and future challenges in epidemiology, surveillance and heaith policy. Memories of the Oswaldo Cruz Institute, 104 Suppl 1: 17-30).
  • the trisaccharide Gal (1,3) Galf (1,4) GlcNAc (alpha-Gal) is a highly expressed immuno-dominant antigen on the surface of T. cruzi, mainly in glycosyl phosphatidyl-inositol anchored mucins.
  • T. cruzi trypomastigote (Acosta-Serrano A et al. 2007. Comparison and evolution of the surface architecture of trypanosomatid parasites In African Trypanosomes: After the Genome, p 319-337.
  • Mottram JC & Acosta-Serrano A (ed) Horizon Scientific Press, Norwich, UK; Travassos LR, Almeida IC 19993.
  • Lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas' disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi (Biochem J 304 (Pt 3): 793-802). Approximately three percent of IgG antibodies from chronically T.
  • cruzi-infected patients recognize the alpha-Gal glycotope, a concentration significantly higher than the approximately 1% of circulating anti-alpha-Gal IgG antibodies in the general human population (Almeida IC , Covas DT, Soussumi LM, Travassos LR.19997.
  • Complement-mediated lysis of Trypanosoma cruzi trypomastigotes by human anti-alpha-galactosyl antibodies Journal of immunology, 146: 2394-2400).
  • bovine serum albumin-linked alpha-galactose-containing glycotopes have been used for serum analysis of chagasic patients, showing promising results as diagnostic antigens for T. cruzi infection (As h mus RA et al. 2013 Potential use of synthetic alpha-galactosyl-containing glycotopes of the parasite Trypanosoma cruzi as diagnostic antigens for Chagas disease Org Biomol Chem, 11: 5579-5583; Schocker NS, Portillo S, Brito CR, Marques AF, Almeida IC, Michael K. 2015.
  • US201462073829P describes a neoglycoconjugate comprising a trisaccharide, Gala (1,3) Gaip (1,4) GlcNAca, bound to a protein carrier such as bovine serum albumin (BSA) or a T cell epitope.
  • BSA bovine serum albumin
  • This document differs from the present invention in that it deals only with the synthesis of different carbohydrates with terminals 1, or 1, 4, for example, linked to the BSA protein, which can be exploited in Chagas disease. These carbohydrates have never been tested extensively, or linked to the bacteriophage Q ⁇ , which can amplify Gal epitope exposure by up to 50-fold.
  • the present invention describes an approach that increases the density and accessibility of the alpha-Gal carbohydrate shown, increasing serum antibody binding specificity and affinity.
  • the VLP viral particles derived from the Q-beta bacteriophage act as a platform for easy production and control.
  • the presentation of several copies of afa-Gal increases the capture of antibodies produced by exposure to T. cruzi, allowing the detection of infection and monitoring the effectiveness of benznidazole treatment, with greater sensitivity.
  • VLP-linked carbohydrate antigens allow the serodiagnosis of Chagas disease for treatment and blood bank purposes.
  • the Q-beta viral particle bound to several copies of the synthetic alpha-Gal trisaccharide may or may not be associated with recombinant proteins such as AdASP-2.
  • Figure 1 represents the steps for the preparation of Q ⁇ (-Gal) 540.
  • the alpha-Gal trisaccharide was covalently linked to the viral particle by copper catalyzed azide-alky cycloaddition.
  • the image on the left represents the crystalline structure of the Q-beta particle.
  • Figure 2 represents the evaluation of antigenicity of different serum pools against different antigens: serum pool of Trypanosoma cruzi (ChP pool) infected patients; healthy donors (non-ChP pool); and sera from patients infected with Leishmania infantum (LV pool). All sera were tested against the following antigens: Q ⁇ (-Gal) 540 1 25 ng / mL; tGPI-Mucine 2 ⁇ g / ml and EpEx at 5 ⁇ g / ml. Q ⁇ (Glu) 540 was used as a control at a concentration of 125 ng / mL. Serum dilutions started at 1/100 followed by 1/200, 1/400 and 1/800. Optical Density (OD) measured at 492 nm.
  • OD Optical Density
  • Figure 3 represents the individual serum tests.
  • the cutoff value (CO 3 for all antigens tested) was defined as the mean antibody titer for negative control antigens plus two standard deviations.
  • Figure 4 represents the recognition of Q ⁇ (-Gal) 540 by serum from untreated patients infected with T. cruzi.
  • the 61 sera from untreated patients were used for Q ⁇ (-Gal) 540 antigen recognition, specifically considering the following properties: A) disease chronicity; B) the parasitemia; C) the age of the patient.
  • Sera were used at 1/800 dilutions and Q ⁇ (-Gal) 540 antigen was used at a concentration of 125 ng / mL.
  • Figure 5 represents the results of treatment follow-up experiments.
  • Figure 6 depicts the protection of Alpha-Gal knock out (KO) animals for alpha-galactosyltransferase using (Q ⁇ -Gal) 540 particles in Trypanosoma cruzi strain Y infected animals.
  • the groups were composed of 7-10 KO animals, immunized weekly (4 doses of 10 ⁇ g / dose) with Q ⁇ -Gal, ASP2 vaccine particles, associated or not, and infected with 10 and 4 Trypanosoma cruzi Y strain. and followed up to 25 weeks.
  • the present invention is directed to a Qf-VLP bacteriophage coupled to several copies of the synthetic alpha-Gal trisaccharide (Gal 1 -3Gal ⁇ f -1 -4GlcNAcR), whether or not associated with recombinant proteins such as Q ⁇ -Gal. AdASP-2, and its use as a vaccine particle.
  • the present invention also describes a method and kit for detection of anti-alpha-Gal antibodies by ELISA for diagnosis of Chagas disease.
  • the method of the present invention enhances the capture of antibodies produced by exposure to T. cruzi, exhibiting high sensitivity and selectivity and enabling detection of infection and monitoring the effectiveness of benznidazole treatment. More specifically, the present invention deals with the bacteriophage-derived Q-beta viral particle bound to an average of 180 to 600 alpha-Gal units.
  • The. exposing a sample to at least one of the Q-beta viral particles bound to multiple copies of the synthetic alpha-Gal trisaccharide (Gal 1 -3Gal ⁇ f -1 -4GlcNAcR), such particles being attached to a solid support or a carrier;
  • step (b) ç. detection of T. cruzi specific antibodies in the sample cited in step (a) using reagents capable of detecting the enzyme or marker cited in step (b).
  • step J the samples used are selected from the group comprising blood, serum, plasma and / or other body fluid.
  • the secondary antibody may be IgG, IgM, IgA, IgE or their subclasses;
  • the protein may be Protein A and / or Protein G;
  • the enzyme which is conjugated to the secondary antibody or protein is selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.
  • the marker is selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent.
  • the enzyme or label detecting reagent is selected from the group comprising any chromogen substrate that is recognized by any of the above enzymes, or any of the above-mentioned markers.
  • the diagnosis kit for Chagas disease is characterized by:
  • the solid support of item a may be selected from the group of materials comprising nitrocellulose, nylon, latex, polypropylene and / or polystyrene. Preferably, it should consist of 96-well microtiter plates, tubes, beads or nitrocellulose and / or nylon papers.
  • the secondary antibody may be IgG, IgM, IgA, IgE and / or their respective subclasses and the protein may be protein A and / or protein G.
  • the enzyme that is conjugated to the secondary antibody or protein may be selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.
  • the marker may be selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and / or chemiluminescent.
  • the reagent for detecting the enzyme or marker of item c may be selected from the group comprising chromogenic substrates that are recognized by any of the above enzymes, or any of the above-mentioned markers.
  • Q-beta bacteriophage-derived viral particles were prepared and purified as described by Fiedler and colleagues (Fiedler JD, Brown SD, Lau JL, Finn MG. 2010. RNA-directed packaging of enzymes within virus-like particles. Angewandte Chemie 49: 9648-9651).
  • the surface of the amino groups of the Q-beta viral particles mainly present on the outside of the particles, was acylated with N-hydroxysuccinimide ester containing an azide group at the end of a short chain (Banerjee D, Liu AP, Voss NR, Schmid SL, Finn MG 2010. Multivalent display and receptor-mediated endocytosis of transferrin on virus-like particles (Chembiochem 11: 1273-1279), as represented by Figure 2.
  • alpha-Gal and glucose were converted to their terminal alkyne derivatives.
  • carbohydrate-alkynes were conjugated to polyvalently exposed azide groups by copper catalyzed azide-alkyne cycloaddition (Hong V, Presolski SI, Ma C, Finn MG. 2009. Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation. Angewandte Chemie 48: 9879-9883).
  • the particles were characterized by molecular filtration chromatography, dynamic light scattering (Wyatt DynaPro), microfluidic electrophoresis (Agilent Bioanalyzer 2100, using Protein 80 chips) and electrospray ionization mass spectrometry. high resolution (HR-ESI-MS, Agilent G6230B). Representative samples were analyzed by transmission electron microscopy and multiangular light scattering (Malvern Viscotec).
  • Serum antigenicity of 161 patients (infected and uninfected by T. cruzi) against immunogenic carbohydrate Gal 1 -3Gal ⁇ f 1 - 4GlcNAcR (alpha-Gal) was verified by ELISA using the VLP Q viral particle - beta chemically modified to average 540 alpha-Gal molecules per capsid.
  • the antigens used in the test were: Q-beta (alpha-Gal) 540, mucin tGPI, and antigens obtained from total epimastigote lysate (EpEx).
  • the antigen concentrations used per experiment and dilutions of serum samples were chosen from preliminary experiments to determine the conditions that would give the highest ChP versus Non-ChP and LV signal rates.
  • the glucose particle Q ⁇ (39) 540 was used as a control antigen.
  • Q ⁇ (-Gal) 540 showed a significantly higher signal against the ChP serum pool, and a significantly lower background noise against the VL serum pool compared to the previously characterized tGPI and EpEx mucin antigens ( De Marchi CR, Di noisya JM, Frasch AC, Amato Grandson V, Almeida IC, Buscaglia CA. 201 1. Evaluation of a recombinant Trypanosoma cruzi mucin-like antigen for serodiagnosis of Chagas' disease Clinical and vaccine immunology: CVI 18: 1850 -1855).
  • ChP subjects who received no treatment were categorized according to their chronic Chagas cardiomyopathy (CCC), parasitemia, and age.
  • Sera collected from each patient were tested for anti-alpha-Gal antibodies using Q ⁇ (-Gal) 540, and the results plotted against each parameter ( Figure 5). No correlation was found in any of these comparisons, indicating that untreated patients have high levels of anti-alpha-Gal antibodies throughout the course of infection.
  • Chagas disease caused by the parasitic protozoan hemoflagellate Trypanosoma cruzi, is one of the most endemic and infectious diseases in Latin America. About 15 million people are currently infected and thousands will die each year due to major clinical complications, particularly cardiomyopathy. Due to increased mobility of the global population, DC has become an emerging infection in the US and Europe, where it is estimated that thousands of individuals are infected with the parasite. Recently, only Benznidazole is commercially available for the treatment of the disease. This medicine has relatively low efficacy in the chronic phase of infection, and has marked toxicity during treatment. In addition, there is no human vaccine to prevent and / or treat CD, despite numerous experimental vaccine studies.
  • Antigens of Glycans remain very poorly exploited as vaccine targets, despite the fact that glycoconjugates (glycolipoproteins and glycolipids) are the major molecules present on the surface of T. cruzi trypomastigotes and contain highly immunogenic epitopes not commonly found in humans.
  • glycoconjugates glycolipoproteins and glycolipids
  • Anti-Gal Antibodies are elicited against epitopes of this trisaccharide present on parasite glycoproteins, both in the acute and chronic phase of human Chagas disease.
  • GalT-KO Knock out mouse model for enzyme 1,3-galactosyltransferase
  • This animal model closely mimics the human humoral immune response against T. cruzi. These animals do not express the Gal epitope on the surface of their cells and tissues, just like old world monkeys and humans.
  • As an immunogen we employ the synthetic trisaccharide Gal 1, 3Gal ⁇ f 1,4,4GlcNAc (Gal) linked to the QB-VLP virus associated with or not to recombinant proteins like AdASP-2.
  • GalT-KO mice were immunized according to the protocol below. In contrast to controls, which died within 12 weeks, all mice immunized with the particles survived up to 50 weeks, also presenting low parasitemia and lower weight loss (endpoint experiment) followed by lethal parasite challenges (strain Y ), Figure 6.

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Abstract

The present invention relates to a Q ∮-VLP bacteriophage coupled to several copies of the synthetic alpha-Gal (Gal 1-3Gal ∮1-4GlcNAcR) trisaccharide, the Q ∮- Gal, optionally associated with recombinant proteins, such as AdASP-2, and the use thereof as a vaccinal particle. The present invention also describes a method and a kit for detecting anti-alpha-Gal antibodies by ELISA, for diagnosing Chagas disease. The method of the present invention increases the capture of antibodies produced by exposure to T. cruzi, with high sensitivity and selectivity, allowing the infection to be detected and the effectiveness of the treatment with benznidazole to be followed up. Figure 1 Ascorbate NA (10 mM) 0.1 M Phosphate buffer pH 7,4

Description

PARTÍCULA VIRAL LIGADA A ANTÍGENO CARBOIDRATO PARA DIAGNÓSTICO DIFERENCIAL DA DOENÇA DE CHAGAS, MÉTODO, KIT,  VIRAL PARTICLE RELATED TO ANTIGEN CARBOHYDRATE FOR DIFFERENTIAL DIAGNOSIS OF CHAGAS DISEASE, METHOD, KIT,
VACINA E USO. VACCINE AND USE.
[001 ] A presente invenção trata de um bacteriógago Qf -VLP acoplado a várias cópias do trissacarídeo sintético alfa-Gal (Gal 1 -3Gal<f 1 -4GlcNAcR), denominado de Q§- Gal, associado ou não a proteínas recombinantes, como a AdASP-2, e seu uso como partícula vacinai. A presente invenção também descreve um método e um kit para detecção de anticorpos anti-alfa-Gal por ELISA, para diagnóstico da doença de Chagas. O método da presente invenção aumenta a captura de anticorpos produzidos por exposição a T. cruzi, apresentando alta sensibilidade e seletividade e possibilitando a detecção da infecção e o acompanhamento da efetividade do tratamento com benznidazol. The present invention is directed to a Qf-VLP bacteriophage coupled to several copies of the synthetic alpha-Gal trisaccharide (Gal 1 -3Gal <f -1 -4GlcNAcR), whether or not associated with recombinant proteins such as Q§-Gal. AdASP-2, and its use as a vaccine particle. The present invention also describes a method and kit for detection of anti-alpha-Gal antibodies by ELISA for diagnosis of Chagas disease. The method of the present invention enhances the capture of antibodies produced by exposure to T. cruzi, exhibiting high sensitivity and selectivity and enabling detection of infection and monitoring the effectiveness of benznidazole treatment.
[002] A doença de Chagas, ou Tripanossomíase americana, é uma doença causada pelo protozoário Trypanosoma cruzi. De acordo com a Organização Mundial da Saúde, estima-se que cerca de 7 milhões de pessoas estejam infectadas no mundo (WHO. 2015. Chagas disease (American trypanosomiasis), on World Health Organization. http:// www.who.int/ mediacentre/ factsheets/ fs340/ en/), com aproximadamente 5,7 milhões de casos na América Latina e aproximadamente 30.000 novos casos por ano (WHO. 2015. Chagas disease in Latin America: an epidemiological update based on 2010 estimates. Wkly Epidemiol Rec, 90:33-43). A doença representa um importante problema de saúde pública e um problema económico, visto que a maioria dos pacientes no estágio crónico da doença se encontra nas idades mais ativas economicamente, além dos altos custos do tratamento e das estratégias de controle do vetor (Moncayo A, Silveira AC. 2009. Current epidemiological trends for Chagas disease in Latin America and future challenges in epidemiology, surveillance and heaith policy. Memórias do Instituto Oswaldo Cruz, 104 Suppl 1 :17-30). Chagas disease, or American Trypanosomiasis, is a disease caused by the protozoan Trypanosoma cruzi. According to the World Health Organization, an estimated 7 million people are infected worldwide (WHO. 2015. Chagas disease (American trypanosomiasis), on World Health Organization. Http: // www.who.int/ mediacentre / factsheets / fs340 / en /), with approximately 5.7 million cases in Latin America and approximately 30,000 new cases per year (WHO. 2015. Chagas disease in Latin America: an epidemiological update based on 2010 estimates. Wkly Epidemiol Rec 90: 33-43). The disease represents a major public health problem and an economic problem, as most patients in the chronic stage of the disease are at the most economically active age, in addition to the high costs of treatment and vector control strategies (Moncayo A, Silveira AC. 2009. Current epidemiological trends for Chagas disease in Latin America and future challenges in epidemiology, surveillance and heaith policy. Memories of the Oswaldo Cruz Institute, 104 Suppl 1: 17-30).
[003] A imigração humana tem contribuído para a expansão da distribuição da doença, com dados sugerindo entre 300.000 e 1 milhão de casos apenas nos Estados Unidos, além de milhares de casos registrados em outros países não- endêmicos (Hotez PJ. 2008. Neglected infections of poverty in the United States of America. PLoS neglected tropical diseases, 2:e256.6; Bern C, Kjos S, Yabsley MJ, Montgomery SP. 201 1 . Trypanosoma cruzi and Chagas' Disease in the United States. Clinicai microbiology reviews, 24:655-681 ; Bern C, Montgomery SP. 2009. An estimate of the burden of Chagas disease in the United States. Clinicai infectious diseases: an official publication of the Infectious Diseases Society of America, 49:e52-54). Em resposta, os países não-endêmicos passaram a solicitar que os imigrantes de ambientes endémicos façam o teste sorológico para anticorpos anti- Trypanossoma cruzi. Essa situação epidemiológica criou a necessidade de melhorias nas metodologias de detecção de exposição a T. cruzi, para um melhor controle da doença.  Human immigration has contributed to the spread of disease distribution, with data suggesting between 300,000 and 1 million cases in the United States alone, as well as thousands of cases reported in other non-endemic countries (Hotez PJ. 2008. Neglected infections of poverty in the United States of America PLoS neglected tropical diseases, 2: e256.6, Bern C, Kjos S, Yabsley MJ, Montgomery SP 201 1. Trypanosoma cruzi and Chagas Disease in the United States. , 24: 655-681, Bern C, Montgomery SP 2009. An estimate of the burden of Chagas disease in the United States Clinical infectious diseases: an official publication of the Infectious Diseases Society of America, 49: e52-54). In response, non-endemic countries now require immigrants from endemic environments to be tested for anti-Trypanosoma cruzi antibodies. This epidemiological situation created the need for improvements in the methodologies for detecting T. cruzi exposure for better disease control.
[004] A maioria dos pacientes com doença de Chagas não apresenta sintomas até que a doença atinja seu estágio crónico. Apenas os pacientes que apresentam sintomas nos estágios iniciais da doença apresentam anticorpos anti- T. cruzi em níveis detectáveis. Porém, no estágio crónico, a presença de patologias severas, como a miocardite e as megasíndromes digestivas, pode dificultar a resposta ao tratamento com benznidazol, que é o único fármaco aprovado pelo FDA para tratamento dessa doença. Este medicamento apresenta eficácia relativamente baixa na fase crónica da infecção, e apresenta acentuada toxicidade durante o tratamento. [005] O diagnóstico da infeção por T. cruzi atualmente requer o uso de no mínimo dois testes convencionais recomendados pela Organização Mundial de Saúde. Os testes sorológicos disponíveis têm produzido um número considerável de diagnósticos questionáveis (Guzman-Gomez D et al. 2015. Highly discordant serology against Trypanosoma cruzi in central Veracruz, México: role of the antigen used for diagnostic. Parasites & vectors, 8:466), por duas razões principais: (a) a maioria dos pacientes chagásicos apresenta baixos níveis de anticorpos anti- T. cruzi, e (b) os antigenos utilizados nos testes são baseados em proteínas purificadas, antigenos recombinantes e peptídeos sintéticos, derivados dos parasitas em seu estágio não-infectante e portanto não encontrados em pacientes infectados (Silveira JF, Umezawa ES, Luquetti AO. 2001 . Chagas disease: recombinant Trypanosoma cruzi antigens for serological diagnosis. Trends Parasitol 17:286-291 ; Caballero ZC et al. 2007. Evaluation of serological tests to identify Trypanosoma cruzi infection in humans and determine cross-reactivity with Trypanosoma rangeli and Leishmania spp. Clin Vaccine Immunol, 14:1045-1049). Além disso, foi demonstrado que o soro de indivíduos com leishmanioses, micoses, ou algumas doenças autoimunes apresentam reação cruzada com preparações de antigenos de T. cruzi (Araujo FG. 1986. Analysis of Trypanosoma cruzi antigens bound by specific antibodies and by antibodies to related trypanosomatids. Infection and immunity, 53:179-185; Schnaidman BB, Yoshida N, Gorin PA, Travassos LR. 1986. Cross-reactive polysaccharides from Trypanosoma cruzi and fungi (especially Dactylium dendroides). The Journal of protozoology, 33:186-191 ). Ademais, esses testes apresentam variações na reprodutibilidade e confiabilidade, que podem ser atribuídas à padronização não-otimizada dos reagentes (da Silveira JF, Umezawa ES, Luquetti AO. 2001 . Chagas disease: recombinant Trypanosoma cruzi antigens for serological diagnosis. Trends in parasitology, 17:286-291 ). Most patients with Chagas disease show no symptoms until the disease reaches its chronic stage. Only patients with symptoms in the early stages of the disease have anti-T. cruzi antibodies at detectable levels. However, at the chronic stage, the presence of severe pathologies such as myocarditis and digestive megaindromes may make it difficult to respond to treatment with benznidazole, which is the only FDA-approved drug to treat this disease. This medicine has relatively low efficacy in the chronic phase of infection, and has marked toxicity during treatment. [005] Diagnosis of T. cruzi infection currently requires the use of at least two conventional tests recommended by the World Health Organization. Available serological tests have produced a considerable number of questionable diagnoses (Guzman-Gomez D et al. 2015. Highly discordant serology against Trypanosoma cruzi in central Veracruz, Mexico: Parasites & vectors, 8: 466), for two main reasons: (a) most chagasic patients have low levels of anti-T antibodies. . cruzi, and (b) the antigens used in the tests are based on purified proteins, recombinant antigens and synthetic peptides, derived from parasites at their non-infective stage and therefore not found in infected patients (Silveira JF, Umezawa ES, Luquetti AO. 2001 Chagas disease: recombinant Trypanosoma cruzi antigens for serological diagnosis Trends Parasitol 17: 286-291 Caballero ZC et al. Evaluation of serological tests to identify Trypanosoma cruzi infection in humans and determine cross-reactivity with Trypanosoma rangeli and Leishmania spp. Clin Vaccine Immunol, 14: 1045-1049). In addition, it has been shown that serum from individuals with leishmaniasis, mycoses, or some autoimmune diseases cross-react with T. cruzi antigen preparations (Araujo FG. 1986. Analysis of Trypanosoma cruzi antigens. trypanosomatids Infection and immunity, 53: 179-185; Schnaidman BB, Yoshida N, Gorin PA, Travassos LR 1986. Cross-reactive polysaccharides from Trypanosoma cruzi and fungi (especially Dactylium dendroides). 191). Moreover, these tests have variations in reproducibility and reliability, which can be attributed to non-optimized reagent standardization (from Silveira JF, Umezawa ES, Luquetti AO. 2001 Chagas disease: recombinant Trypanosoma cruzi antigens for serological diagnosis. Trends in parasitology, 17: 286-291).
[006] O trissacarídeo Gal (1 ,3)Galf (1 ,4)GlcNAc (alfa-Gal) é um antígeno imuno-dominante altamente expresso na superfície de T. cruzi, principalmente em mucinas ancoradas em glicosil-fosfatidil-inositol da forma infectante tripomastigota de T. cruzi (Acosta-Serrano A et al. 2007. Comparison and evolution of the surface architecture of trypanosomatid parasites In African Trypanosomes: After the Genome, p 319-337. In Barry J D MR, Mottram JC & Acosta-Serrano A (ed). Horizon Scientific Press, Norwich, UK; Travassos LR, Almeida IC. 1 993. Carbohydrate immunity in American trypanosomiasis. Springer Seminars in Immunopathology, 15:183-204; Almeida IC, Ferguson MA, Schenkman S, Travassos LR. 1994. Lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas' disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi. Biochem J 304 ( Pt 3):793-802). Aproximadamente três por cento dos anticorpos IgG de pacientes cronicamente infectados por T. cruzi reconhecem o glicotopo alfa-Gal, uma concentração significativamente mais alta do que os aproximadamente 1 % de anticorpos IgG anti-alfa-Gal circulantes na população humana em geral (Almeida IC, Covas DT, Soussumi LM, Travassos LR. 1 997. A highly sensitive and specific chemiluminescent enzyme-linked immunosorbent assay for diagnosis of active Trypanosoma cruzi infection. Transfusion, 37:850-857; Almeida IC, Milani SR, Gorin PA, Travassos LR. 1 991 . Complement-mediated lysis of Trypanosoma cruzi trypomastigotes by human anti-alpha-galactosyl antibodies. Journal of immunology, 146:2394-2400). The trisaccharide Gal (1,3) Galf (1,4) GlcNAc (alpha-Gal) is a highly expressed immuno-dominant antigen on the surface of T. cruzi, mainly in glycosyl phosphatidyl-inositol anchored mucins. T. cruzi trypomastigote (Acosta-Serrano A et al. 2007. Comparison and evolution of the surface architecture of trypanosomatid parasites In African Trypanosomes: After the Genome, p 319-337. In Barry JD MR, Mottram JC & Acosta-Serrano A (ed) Horizon Scientific Press, Norwich, UK; Travassos LR, Almeida IC 19993. Carbohydrate immunity in American trypanosomiasis, Springer Seminars in Immunopathology, 15: 183-204; Almeida IC, Ferguson MA, Schenkman S, Travassos LR 1994. Lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas' disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi (Biochem J 304 (Pt 3): 793-802). Approximately three percent of IgG antibodies from chronically T. cruzi-infected patients recognize the alpha-Gal glycotope, a concentration significantly higher than the approximately 1% of circulating anti-alpha-Gal IgG antibodies in the general human population (Almeida IC , Covas DT, Soussumi LM, Travassos LR.19997. A highly sensitive and specific chemiluminescent enzyme-linked immunosorbent assay for diagnosis of active Trypanosoma cruzi infection Transfusion, 37: 850-857; Almeida IC, Milani SR, Gorin PA, Travassos LR 1991. Complement-mediated lysis of Trypanosoma cruzi trypomastigotes by human anti-alpha-galactosyl antibodies (Journal of immunology, 146: 2394-2400).
[007] A avaliação do tratamento da doença de Chagas na fase crónica é controversa e difícil de validar, visto que não há critérios confiáveis de sucesso durante esse estágio. Na tentativa de detectar anticorpos para o tratamento da doença de Chagas, dois estudos utilizaram mucina purificada de tripomastigotas, contendo o antígeno alfa-Gal, para avaliar os níveis de anticorpos após tratamento com benznidazol, e correlacionaram os resultados com a progressão da doença. No entanto, esta abordagem é limitada, devido, em parte, às dificuldades de se extrair mucina (Andrade AL et al. 2004. Short report: benznidazole efficacy among Trypanosoma craz/-infected adolescents after a six-year follow-up. The American journal of tropical medicine and hygiene 71 :594-597; de Andrade AL et al. 1 996. Randomised trial of efficacy of benznidazole in treatment of early Trypanosoma cruzi infection. Lancet, 348:1407-141 3). Evaluation of the treatment of Chagas disease in the chronic phase is controversial and difficult to validate as there are no reliable criteria for success. during this stage. In an attempt to detect antibodies for the treatment of Chagas disease, two studies used purified trypomastigote mucin containing alpha-Gal antigen to evaluate antibody levels after benznidazole treatment and correlated the results with disease progression. However, this approach is limited, due in part to the difficulties in extracting mucin (Andrade AL et al. 2004. Short report: benznidazole efficacy among Trypanosoma craz / infected adolescents after a six-year follow-up. The American Journal of Tropical Medicine and Hygiene 71: 594-597; by Andrade AL et al., 1996. Randomized trial of efficacy of benznidazole in treatment of early Trypanosoma cruzi infection. Lancet, 348: 1407-141 3).
[008] Recentemente, glicotopos sintéticos contendo alfa-galactose ligados à soroalbumina bovina (BSA) foram usados para a análise de soros de pacientes chagásicos, apresentando resultados promissores como antígenos diagnósticos para infecção por T. cruzi (As h mus RA et al. 2013. Potential use of synthetic alpha-galactosyl-containing glycotopes of the parasite Trypanosoma cruzi as diagnostic antigens for Chagas disease. Org Biomol Chem, 1 1 :5579-5583; Schocker NS, Portillo S, Brito CR, Marques AF, Almeida IC, Michael K. 2015. Synthesis of Galalpha(1 ,3)Galbeta(1 ,4)GlcNAcalpha-, Galbeta(1 ,4)GlcNAcalpha-, and GIcNAcalpha-containing neoglycoproteins and their immunological evaluation in the context of Chagas disease. Glycobiology).  Recently, bovine serum albumin-linked alpha-galactose-containing glycotopes (BSA) have been used for serum analysis of chagasic patients, showing promising results as diagnostic antigens for T. cruzi infection (As h mus RA et al. 2013 Potential use of synthetic alpha-galactosyl-containing glycotopes of the parasite Trypanosoma cruzi as diagnostic antigens for Chagas disease Org Biomol Chem, 11: 5579-5583; Schocker NS, Portillo S, Brito CR, Marques AF, Almeida IC, Michael K. 2015. Synthesis of Galalpha (1,3) Galbeta (1,4) GlcNAcalpha-, Galbeta (1,4) GlcNAcalpha-, and GIcNAcalpha-containing neoglycoproteins and their immunological evaluation in the context of Chagas disease. Glycobiology).
[009] O documento de patente US201462073829P descreve um neoglicoconjugado compreendendo um trissacarídeo, Gala(l ,3)Gaip(l ,4)GlcNAca, ligado a um carreador protéico, como a soroalbumina bovina (BSA) ou um epítopo de célula T. Esse documento se difere da presente invenção porque trata apenas da síntese de diferentes carboidratos com terminais 1 , ou 1 ,4 por exemplo, ligados à proteína BSA, que podem ser explorados na doença de Chagas. Esses carboidratos em nenhum momento foram testados amplamente, ou ligados ao bacteriofago Q§ , o qual pode amplificar a exposição dos epitopos Gal em cerca de 50 vezes. US201462073829P describes a neoglycoconjugate comprising a trisaccharide, Gala (1,3) Gaip (1,4) GlcNAca, bound to a protein carrier such as bovine serum albumin (BSA) or a T cell epitope. This document differs from the present invention in that it deals only with the synthesis of different carbohydrates with terminals 1, or 1, 4, for example, linked to the BSA protein, which can be exploited in Chagas disease. These carbohydrates have never been tested extensively, or linked to the bacteriophage Q§, which can amplify Gal epitope exposure by up to 50-fold.
[010] A presente invenção descreve uma abordagem que aumenta a densidade e a acessibilidade do carboidrato alfa-Gal apresentado, aumentando a especificidade e a afinidade de ligação a anticorpos do soro. As partículas virais VLP derivadas do bacteriofago Q-beta atuam como uma plataforma de fácil produção e controle. A apresentação de várias cópias de afa-Gal aumenta a captura de anticorpos produzidos por exposição a T. cruzi, possibilitando a detecção da infecção e o acompanhamento da efetividade do tratamento com benznidazol, com maior sensibilidade. Os antígenos carboidratos ligados a VLPs permitem o sorodiagnóstico da doença de Chagas para propósitos de tratamentos e de bancos de sangue. A partícula virai Q- beta ligada a várias cópias do trissacarídeo sintético alfa-Gal (Gal 1 -3Gal<f 1 - 4GlcNAcR), pode estar associada ou não a proteínas recombinantes, como a AdASP-2. [010] The present invention describes an approach that increases the density and accessibility of the alpha-Gal carbohydrate shown, increasing serum antibody binding specificity and affinity. The VLP viral particles derived from the Q-beta bacteriophage act as a platform for easy production and control. The presentation of several copies of afa-Gal increases the capture of antibodies produced by exposure to T. cruzi, allowing the detection of infection and monitoring the effectiveness of benznidazole treatment, with greater sensitivity. VLP-linked carbohydrate antigens allow the serodiagnosis of Chagas disease for treatment and blood bank purposes. The Q-beta viral particle bound to several copies of the synthetic alpha-Gal trisaccharide (Gal 1 -3Gal <f 1-4GlcNAcR) may or may not be associated with recombinant proteins such as AdASP-2.
[01 1 ] No estado da técnica não foi encontrada tecnologia semelhante à descrita na presente invenção.  In the prior art no technology similar to that described in the present invention has been found.
BREVE DESCRIÇÃO DAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
[012] A figura 1 representa os passos para a preparação de Q§ ( -Gal)540. O trissacarídeo alfa-Gal foi ligado covalentemente à partícula virai por cicloadição azida-alquino catalisada por cobre. A imagem na esquerda representa a estrutura cristalina da partícula Q-beta.  Figure 1 represents the steps for the preparation of Q§ (-Gal) 540. The alpha-Gal trisaccharide was covalently linked to the viral particle by copper catalyzed azide-alky cycloaddition. The image on the left represents the crystalline structure of the Q-beta particle.
[013] A figura 2 representa a avaliação da antigenicidade de diferentes pools de soros contra diferentes antígenos: pool de soros de pacientes infectados com Trypanosoma cruzi (ChP pool); doadores saudáveis (não-ChP pool); e soros de pacientes infectados por Leishmania infantum (LV pool). Todos os soros foram testados contra os seguintes antígenos: Q§ ( -Gal)540 1 25 ng/mL; tGPI-Mucina 2^g/mL e EpEx a 5μg/ mL. Q§ (Glu)540 foi usado como controle na concentração de 125 ng/mL. As diluições dos soros se iniciaram em 1 /1 00 seguidas por 1 /200, 1 /400 e 1 /800. Densidade Óptica (D. O.) medida em 492 nm. [013] Figure 2 represents the evaluation of antigenicity of different serum pools against different antigens: serum pool of Trypanosoma cruzi (ChP pool) infected patients; healthy donors (non-ChP pool); and sera from patients infected with Leishmania infantum (LV pool). All sera were tested against the following antigens: Q§ (-Gal) 540 1 25 ng / mL; tGPI-Mucine 2 µg / ml and EpEx at 5 µg / ml. Q§ (Glu) 540 was used as a control at a concentration of 125 ng / mL. Serum dilutions started at 1/100 followed by 1/200, 1/400 and 1/800. Optical Density (OD) measured at 492 nm.
[014] A figura 3 representa os testes de soros individuais. A reatividade dos soros coletados de pacientes chagásicos não tratados (ChP) infectados com T. cruzi (n=61 ), de pacientes não-chagásicos ou doadores saudáveis (não- ChP; n=45) e infectados com Leismania infantum (LV; n=46) foram avaliadas contra os seguintes antígenos: A) lisado total de epimastigota (EpEx), 5 μg/mL; B) Mucina tGPI purificada, 2,5 Mg/mL; e C) Antígeno carboidrato Q§ ( - Gal)540, 1 25 ng/mL; D) Comparação da Especificidade (S=TN/TN+FP) e da Sensibilidade (Se= TP/TP+FN) entre os antígenos descritos acima. Os resultados foram normalizados pela equação: a + (x-A)*(b-a)/B-A, onde A= valor mínimo obtido; B = valor máximo obtido; a = 1 e b=9. O valor de corte (CO=3 para todos os antígenos testados) foi definido como a média do título de anticorpos para antígenos controle negativos mais dois desvios padrão. [014] Figure 3 represents the individual serum tests. The reactivity of sera collected from untreated chagasic patients (ChP) infected with T. cruzi (n = 61), non-chagasic patients or healthy donors (non-ChP; n = 45) and infected with Leismania infantum (LV; n = 46) were evaluated against the following antigens: A) total epimastigote lysate (EpEx), 5 μg / mL; B) Purified tGPI Mucine, 2.5 Mg / mL; and C) Q§ (-Gal) 540 carbohydrate antigen, 125 ng / mL; D) Comparison of Specificity (S = TN / TN + FP) and Sensitivity (Se = TP / TP + FN) between the antigens described above. Results were normalized by the equation: a + (xA) * (ba) / BA, where A = minimum value obtained; B = maximum value obtained; a = 1 and b = 9. The cutoff value (CO = 3 for all antigens tested) was defined as the mean antibody titer for negative control antigens plus two standard deviations.
[01 5] A figura 4 representa o reconhecimento de Q§ ( -Gal)540 pelo soro de pacientes não tratados infectados por T. cruzi. Os 61 soros de pacientes não tratados foram usados para reconhecimento do antígeno Q§ ( -Gal)540, considerando-se, especificamente as seguintes propriedades: A) a cronicidade da doença; B) a parasitemia; C) a idade do paciente. Os soros foram usados nas diluições de 1 /800 e o antígeno Q§ ( -Gal)540 foi usado na concentração de 125 ng/mL. Figure 4 represents the recognition of Q§ (-Gal) 540 by serum from untreated patients infected with T. cruzi. The 61 sera from untreated patients were used for Q§ (-Gal) 540 antigen recognition, specifically considering the following properties: A) disease chronicity; B) the parasitemia; C) the age of the patient. Sera were used at 1/800 dilutions and Q§ (-Gal) 540 antigen was used at a concentration of 125 ng / mL.
[01 6] A figura 5 representa os resultados dos experimentos de acompanhamento de tratamento. A) O soro de dois pacientes foi coletado 1 , 6, 12, 18 θ 24 meses após tratamento com benznidazol (círculo aberto). O quadrado aberto indica os resultados de dois pacientes não tratados, com soros coletados nos mesmos intervalos de tempo. B) Reatividade do soro de 5 pacientes infectados e tratados coletados em diferentes tempos (em meses) após tratamento com benznidazol. Os pacientes foram identificados de acordo com a data da coleta do soro: TP 003; TP 012; TP 1 16; TP 019; TP 050. A densidade ótica foi medida a 492 nm. [01 6] Figure 5 represents the results of treatment follow-up experiments. A) The serum from two patients was collected 1, 6, 12, 18 θ 24 months after benznidazole treatment (open circle). The open square indicates the results of two untreated patients with sera collected at the same time intervals. B) Serum reactivity of 5 infected and treated patients collected at different times (in months) after treatment with benznidazole. Patients were identified according to the date of serum collection: TP 003; TP 012; TP 1 16; TP 019; TP 050. Optical density was measured at 492 nm.
[017] A figura 6 representa a proteção de animais Alpha-Gal knock out (KO) para alpha-galactosiltransferase usando partículas de (Q§- Gal)540 em animais infectados com Trypanosoma cruzi cepa Y. A) Peso corporal dos animais KO; B) Parasitemia; C) Sobrevivência. Os grupos foram compostos de 7-10 animais KO, imunizados semanalmente (4 doses de 10 μg/dose) com as partículas vacinais Q§- Gal, ASP2, associadas ou não, e infectados com 10 e 4 parasitos de cepa Y de Trypanosoma cruzi e acompados por até 25 semanas.  Figure 6 depicts the protection of Alpha-Gal knock out (KO) animals for alpha-galactosyltransferase using (Q§-Gal) 540 particles in Trypanosoma cruzi strain Y infected animals. A) Body weight of KO animals; B) Parasitemia; C) Survival. The groups were composed of 7-10 KO animals, immunized weekly (4 doses of 10 μg / dose) with Q§-Gal, ASP2 vaccine particles, associated or not, and infected with 10 and 4 Trypanosoma cruzi Y strain. and followed up to 25 weeks.
DESCRIÇÃO DETALHADA DA TECNOLOGIA  DETAILED DESCRIPTION OF TECHNOLOGY
[018] A presente invenção trata de um bacteriógago Qf -VLP acoplado a várias cópias do trissacarídeo sintético alfa-Gal (Gal 1 -3Gal<f 1 -4GlcNAcR), denominado de Q§- Gal, associado ou não a proteínas recombinantes, como a AdASP-2, e seu uso como partícula vacinai. A presente invenção também descreve um método e um kit para detecção de anticorpos anti-alfa-Gal por ELISA, para diagnóstico da doença de Chagas. O método da presente invenção aumenta a captura de anticorpos produzidos por exposição a T. cruzi, apresentando alta sensibilidade e seletividade e possibilitando a detecção da infecção e o acompanhamento da efetividade do tratamento com benznidazol. [019] Mais especificamente, a presente invenção trata da partícula virai derivada do bacteriófago Q-beta ligada a uma média de 180 a 600 unidades de alfa-Gal. [018] The present invention is directed to a Qf-VLP bacteriophage coupled to several copies of the synthetic alpha-Gal trisaccharide (Gal 1 -3Gal <f -1 -4GlcNAcR), whether or not associated with recombinant proteins such as Q§-Gal. AdASP-2, and its use as a vaccine particle. The present invention also describes a method and kit for detection of anti-alpha-Gal antibodies by ELISA for diagnosis of Chagas disease. The method of the present invention enhances the capture of antibodies produced by exposure to T. cruzi, exhibiting high sensitivity and selectivity and enabling detection of infection and monitoring the effectiveness of benznidazole treatment. More specifically, the present invention deals with the bacteriophage-derived Q-beta viral particle bound to an average of 180 to 600 alpha-Gal units.
[020] O método proposto para o diagnóstico da doença de Chagas caracteriza-se por compreender as seguintes etapas:  [020] The proposed method for the diagnosis of Chagas disease is characterized by the following steps:
a. exposição de uma amostra a pelo menos uma das partícula virais Q-beta ligadas a várias cópias do trissacarídeo sintético alfa-Gal (Gal 1 -3Gal<f 1 -4GlcNAcR), estando tais partículas ligadas a um suporte sólido ou a um carreador;  The. exposing a sample to at least one of the Q-beta viral particles bound to multiple copies of the synthetic alpha-Gal trisaccharide (Gal 1 -3Gal <f -1 -4GlcNAcR), such particles being attached to a solid support or a carrier;
b. adição de um anticorpo secundário ou uma proteína, que estejam conjugados a uma enzima ou a um marcador e que se liguem aos anticorpos da amostra da etapa (a);  B. addition of a secondary antibody or protein which is conjugated to an enzyme or label and which binds to the antibodies of the sample from step (a);
c. detecção dos anticorpos específicos para T. cruzi na amostra citada na etapa (a), utilizando-se reagentes capazes de detectar a enzima ou o marcador citados na etapa (b).  ç. detection of T. cruzi specific antibodies in the sample cited in step (a) using reagents capable of detecting the enzyme or marker cited in step (b).
[021 ] Na etapa aJ as amostras utilizadas são selecionadas do grupo compreendendo sangue, soro, plasma e/ou outro fluído corporal. In step J the samples used are selected from the group comprising blood, serum, plasma and / or other body fluid.
[022] Na etapa o anticorpo secundário pode ser IgG, IgM, IgA, IgE ou respectivas subclasses; a proteína pode ser a Proteína A e/ou a Proteína G; e a enzima que está conjugada ao anticorpo secundário ou à proteína é selecionada do grupo compreendendo peroxidase, fosfatase alcalina, beta- galactosidase, urease, xantina oxidase, glicose oxidase e penicilinase. Já o marcador é selecionado do grupo compreendendo enzimas, radioisótopos, biotina, cromóforos, fluoróforos e quimioluminescentes. Por fim, na etapa c_, o reagente para detectar a enzima ou o marcador é selecionado do grupo compreendendo qualquer substrato cromógeno que seja reconhecido por alguma das enzimas supracitadas, ou algum dos marcadores supracitados. [023] O kit para o diagnóstico da Doença de Chagas caracteriza-se por compreender: In the step the secondary antibody may be IgG, IgM, IgA, IgE or their subclasses; the protein may be Protein A and / or Protein G; and the enzyme which is conjugated to the secondary antibody or protein is selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase. The marker is selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent. Finally, in step c, the enzyme or label detecting reagent is selected from the group comprising any chromogen substrate that is recognized by any of the above enzymes, or any of the above-mentioned markers. [023] The diagnosis kit for Chagas disease is characterized by:
a. suporte sólido ou carreador contendo pelo menos uma das partículas virais Q-beta ligadas a várias cópias do trissacarídeo sintético alfa-Gal (Gal 1 -3Galf 1 -4GlcNAcR);  The. solid or carrier support containing at least one of the Q-beta viral particles bound to multiple copies of the synthetic alpha-Gal trisaccharide (Gal 1 -3Galf 1 -4GlcNAcR);
b. anticorpo secundário ou uma proteína, conjugados a uma enzima ou a um marcador;  B. secondary antibody or protein, conjugated to an enzyme or a marker;
c. reagente para detectar a enzima ou marcador.  ç. reagent to detect the enzyme or label.
[024] O suporte sólido do item a. pode ser selecionado do grupo de materiais compreendendo nitrocelulose, nylon, látex, polipropileno e/ou poliestireno. Preferencialmente, deve consistir em placas de microtitulação de 96 poços, tubos, esferas ou papéis de nitrocelulose e/ou nylon. [024] The solid support of item a . may be selected from the group of materials comprising nitrocellulose, nylon, latex, polypropylene and / or polystyrene. Preferably, it should consist of 96-well microtiter plates, tubes, beads or nitrocellulose and / or nylon papers.
[025] No item "b_ o anticorpo secundário pode ser IgG, IgM, IgA, IgE e/ou suas respectivas subclasses e a proteína pode ser a proteína A e/ou proteína G. In " b_ " the secondary antibody may be IgG, IgM, IgA, IgE and / or their respective subclasses and the protein may be protein A and / or protein G.
[026] Para os itens ¾_ e c_, a enzima que está conjugada ao anticorpo secundário ou proteína pode ser selecionada do grupo compreendendo peroxidase, fosfatase alcalina, beta-galactosidase, urease, xantina oxidase, glicose oxidase e penicilinase. Já o marcador pode ser selecionado do grupo compreendendo enzimas, radioisótopos, biotina, cromóforos, fluoróforos e/ou quimioluminescentes. For items e and c_, the enzyme that is conjugated to the secondary antibody or protein may be selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase. The marker may be selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and / or chemiluminescent.
[027] O reagente para detectar a enzima ou o marcador do item c_ pode ser selecionado do grupo compreendendo substratos cromógenos que sejam reconhecidos por alguma das enzimas supracitadas, ou algum dos marcadores supracitados.  The reagent for detecting the enzyme or marker of item c may be selected from the group comprising chromogenic substrates that are recognized by any of the above enzymes, or any of the above-mentioned markers.
[028] A presente invenção pode ser mais bem compreendida através dos exemplos que se seguem, não limitantes. EXEMPLO 1 " SÍNTESE DAS PARTÍCULAS VIRAIS Q-BETA, LIGADAS A ALFA-GAL The present invention may be better understood by the following non-limiting examples. EXAMPLE 1 " SUMMARY OF Q-BETA VIRAL PARTICULARS LINKED TO ALPHA
[029] As partículas virais derivadas do bacteriófago Q-beta foram preparadas e purificadas como descrito por Fiedler e colaboradores (Fiedler JD, Brown SD, Lau JL, Finn MG. 2010. RNA-directed packaging of enzymes within virus- like particles. Angewandte Chemie 49:9648-9651 ). Em suma, a superfície dos grupos amino das partículas virais Q-beta, principalmente presentes no exterior das partículas, foi acilada com éster de N-hidroxisuccinimida contendo um grupo azida ao final de uma cadeia curta (Banerjee D, Liu AP, Voss NR, Schmid SL, Finn MG. 2010. Multivalent display and receptor-mediated endocytosis of transferrin on virus-like particles. Chembiochem 1 1 :1273-1279), como representado pela Figura 2.  [029] Q-beta bacteriophage-derived viral particles were prepared and purified as described by Fiedler and colleagues (Fiedler JD, Brown SD, Lau JL, Finn MG. 2010. RNA-directed packaging of enzymes within virus-like particles. Angewandte Chemie 49: 9648-9651). In summary, the surface of the amino groups of the Q-beta viral particles, mainly present on the outside of the particles, was acylated with N-hydroxysuccinimide ester containing an azide group at the end of a short chain (Banerjee D, Liu AP, Voss NR, Schmid SL, Finn MG 2010. Multivalent display and receptor-mediated endocytosis of transferrin on virus-like particles (Chembiochem 11: 1273-1279), as represented by Figure 2.
[030] Após a síntese, alfa-Gal e glicose foram convertidas em seus derivados alquino terminais. Estes carboidratos-alquino foram conjugados com grupos azida polivalentemente expostos, por cicloadição azida-alquino, catalisada por cobre (Hong V, Presolski SI, Ma C, Finn MG. 2009. Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation. Angewandte Chemie 48:9879-9883).  Following synthesis, alpha-Gal and glucose were converted to their terminal alkyne derivatives. These carbohydrate-alkynes were conjugated to polyvalently exposed azide groups by copper catalyzed azide-alkyne cycloaddition (Hong V, Presolski SI, Ma C, Finn MG. 2009. Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation. Angewandte Chemie 48: 9879-9883).
[031 ] Após purificação por ultracentrifugação em gradiente de sacarose, as partículas foram caracterizadas por cromatografia de filtração molecular, espalhamento dinâmico de luz (Wyatt DynaPro), eletroforese microfluídica (Agilent Bioanalyzer 2100, using Protein 80 chips) e espectrometria de massas por ionização eletrospray de alta resolução (HR-ESI-MS, Agilent G6230B). As amostras representativas foram analisadas por microscopia eletrônica de transmissão e espalhamento de luz multiangular (Malvern Viscotec).  Following purification by sucrose gradient ultracentrifugation, the particles were characterized by molecular filtration chromatography, dynamic light scattering (Wyatt DynaPro), microfluidic electrophoresis (Agilent Bioanalyzer 2100, using Protein 80 chips) and electrospray ionization mass spectrometry. high resolution (HR-ESI-MS, Agilent G6230B). Representative samples were analyzed by transmission electron microscopy and multiangular light scattering (Malvern Viscotec).
[032] Em todos os casos, as propriedades padrão de tamanho e composição foram observadas, com as partículas apresentando uma distribuição de tamanho estreita e uma alta pureza de proteínas (menos de 5% de impurezas proteicas foram detectados). As concentrações de proteína em solução foram medidas com o reagente Coomasie Plus (Pierce), utilizando padrão de BSA. [032] In all cases, standard size and composition properties were observed, with the particles exhibiting a distribution of narrow size and high protein purity (less than 5% protein impurities were detected). Protein concentrations in solution were measured with Coomasie Plus reagent (Pierce) using BSA standard.
[033] Foram detectadas em média 540 unidades de alfa-Gal por partícula, através de análise por espectrometria de massas, com as estruturas dos capsídeos permanecendo intactas e altamente estáveis após armazenamento (Figura 2). 540 alpha-Gal units per particle were detected on average by mass spectrometric analysis, with the capsid structures remaining intact and highly stable after storage (Figure 2).
EXEMPLO 2 ■ TESTE DA ESPECIFICIDADE DO SORODIAGNÓSTICO POR ELISA EXAMPLE 2 ■ ELISA SORODIAGNOSIS SPECIFICITY TEST
[034] A antigenicidade dos soros de 161 pacientes (infectados e não- infectados por T. cruzi) contra o carboidrato imunogênico Gal 1 -3Gal<f 1 - 4GlcNAcR (alfa-Gal) foi verificada por ELISA, usando a partícula virai VLP Q- beta quimicamente modificada para apresentar uma média de 540 moléculas de alfa-Gal por capsídeo. O experimento foi conduzido com um pool de amostras de pacientes chagásicos não tratados, como controle positivo, designado ChP (n=20); um pool de amostras de doadores saudáveis não- chagásicos, como controle negativo, designado não-ChP (n=20); e um pool de amostras de pacientes com leishmaniose visceral, designado LV (n=20). Os antígenos utilizados no teste foram: Q-beta(alfa-Gal)540, mucina tGPI, e antígenos obtidos de lisado total de epimastigotas (EpEx). As concentrações de antígeno utilizadas por experimento e as diluições das amostras de soro foram escolhidas a partir de experimentos preliminares para determinar as condições que dariam as maiores taxas de sinais de ChP versus Não-ChP e LV. A partícula apresentando glicose Q§ (39)540 foi utilizada como antígeno controle.  Serum antigenicity of 161 patients (infected and uninfected by T. cruzi) against immunogenic carbohydrate Gal 1 -3Gal <f 1 - 4GlcNAcR (alpha-Gal) was verified by ELISA using the VLP Q viral particle - beta chemically modified to average 540 alpha-Gal molecules per capsid. The experiment was conducted with a pool of untreated chagasic patient samples as positive control, designated ChP (n = 20); a pool of healthy non-chagasic donor samples, as negative control, designated non-ChP (n = 20); and a sample pool of patients with visceral leishmaniasis, designated LV (n = 20). The antigens used in the test were: Q-beta (alpha-Gal) 540, mucin tGPI, and antigens obtained from total epimastigote lysate (EpEx). The antigen concentrations used per experiment and dilutions of serum samples were chosen from preliminary experiments to determine the conditions that would give the highest ChP versus Non-ChP and LV signal rates. The glucose particle Q§ (39) 540 was used as a control antigen.
[035] Para Q§ ( -Gal)540, o ponto de antigenicidade máxima foi atingido na concentração de 125 ng/mL (6,25 ng/poço). Para a mucina tGPI e EpEx, os melhores perfis foram alcançados nas concentrações de 2,5 Mg/mL (125 ng/poço) e 5 Mg/mL (250 ng/poço), respectivamente. Embora tenhamos testado um total de sete diluições, de 1 :100 a 1 :6400, para os três pools de amostra de soro, as maiores taxas para os antígenos acima foram obtidas quando os soros ChP foram diluídos entre 1 /100 e 1 /800. As respostas para os três antígenos com os soros não-ChP e LV foram bem mais baixas em comparação com aquelas observadas para soros ChP. A resposta sorológica para Q§ (39)540 foi insignificante quando comparada com Q§( -Gal)540, sugerindo a especificidade da alfa-Gal terminal na resposta imune de pacientes infectados (Figura 2). For Q§ (-Gal) 540, the maximum antigenicity point was reached at the concentration of 125 ng / mL (6.25 ng / well). For tGPI and EpEx mucin, the better profiles were achieved at concentrations of 2.5 Mg / mL (125 ng / well) and 5 Mg / mL (250 ng / well), respectively. Although we tested a total of seven dilutions, from 1: 100 to 1: 6400, for the three serum sample pools, the highest rates for the above antigens were obtained when ChP sera were diluted between 1/100 and 1/800. . Responses to the three antigens with non-ChP and LV sera were much lower compared to those observed for ChP sera. The serological response to Q§ (39) 540 was negligible when compared to Q§ (-Gal) 540, suggesting the specificity of terminal alpha-Gal in the immune response of infected patients (Figure 2).
[036] Q§( -Gal)540 apresentou um sinal significativamente maior contra o pool de soro ChP, e um barulho de fundo significativamente menor contra o pool de soro VL, comparado com os antígenos padrões mucina tGPI e EpEx, já caracterizados anteriormente (De Marchi CR, Di Noia JM, Frasch AC, Amato Neto V, Almeida IC, Buscaglia CA. 201 1 . Evaluation of a recombinant Trypanosoma cruzi mucin-like antigen for serodiagnosis of Chagas' disease. Clinicai and vaccine immunology : CVI 18:1850-1855).  [036] Q§ (-Gal) 540 showed a significantly higher signal against the ChP serum pool, and a significantly lower background noise against the VL serum pool compared to the previously characterized tGPI and EpEx mucin antigens ( De Marchi CR, Di Noia JM, Frasch AC, Amato Grandson V, Almeida IC, Buscaglia CA. 201 1. Evaluation of a recombinant Trypanosoma cruzi mucin-like antigen for serodiagnosis of Chagas' disease Clinical and vaccine immunology: CVI 18: 1850 -1855).
[037] Outros experimentos foram realizados na diluição padrão de 1/800, para minimizar a ligação não-específica.  Other experiments were performed at standard dilution of 1/800 to minimize non-specific binding.
[038] Em seguida, foram realizadas análises de ELISA em amostras de soro individuais, como mostrado na Figura 3A-C. Os soros de indivíduos pertencentes aos grupos ChP, não-ChP e LV, e aqueles que não receberam nenhum tipo de tratamento, reagiram aos três antígenos com perfis semelhantes, como esperado, mas com sensibilidades diferentes. Os soros de pacientes ChP não tratados mostraram uma maior resposta a Q§ ( -Gal)540 e produziram os melhores resultados de reação não-cruzada. Da mesma forma, usando Q§ ( -Gal)540, a diferença entre as titulações de soro dos indivíduos ChP não tratados menos reativos e as dos indivíduos não tratados dos grupos LV e não-ChP foi maior que as mesmas diferenças observadas quando os antígenos mucina tGPI e EpEx foram usados. Como mostrado na Figura 3D, os valores de especificidade e sensibilidade foram 96,7% e 77% para EpEx, 95,6% e 93,6% para mucina tGPI, e 100% em ambas medidas para Q§ ( - Gal)540. ELISA analyzes were then performed on individual serum samples as shown in Figure 3A-C. Sera from individuals belonging to the ChP, non-ChP and LV groups, and those who received no treatment, reacted to the three antigens with similar profiles, as expected, but with different sensitivities. Sera from untreated ChP patients showed a higher response to Q§ (-Gal) 540 and produced the best uncrossed reaction results. Similarly, using Q§ (-Gal) 540, the difference between serum titrations of individuals Less reactive untreated ChP and untreated individuals from the LV and non-ChP groups were greater than the same differences observed when the tGPI and EpEx mucin antigens were used. As shown in Figure 3D, specificity and sensitivity values were 96.7% and 77% for EpEx, 95.6% and 93.6% for tGPI mucin, and 100% for both Q§ (- Gal) 540 measurements. .
EXEMPLO 3 " ACOMPANHAMENTO DO TRATAMENTO DE PACIENTES CHAGÁSICOS EXAMPLE 3 " FOLLOW UP ON CHAGASTIC PATIENT TREATMENT
[039] Os indivíduos ChP que não receberam nenhum tipo de tratamento foram categorizados de acordo com sua cardiomiopatia de Chagas crónica (CCC), parasitemia, e idade. Os soros coletados de cada um dos pacientes foram testados para anticorpos anti-alfa-Gal usando Q§ ( -Gal)540, e os resultados foram plotados contra cada um dos parâmetros (Figura 5). Nenhuma correlação foi encontrada em nenhuma dessas comparações, indicando que os pacientes não tratados apresentam altos níveis de anticorpos anti-alfa-Gal durante todo o curso da infecção.  [039] ChP subjects who received no treatment were categorized according to their chronic Chagas cardiomyopathy (CCC), parasitemia, and age. Sera collected from each patient were tested for anti-alpha-Gal antibodies using Q§ (-Gal) 540, and the results plotted against each parameter (Figure 5). No correlation was found in any of these comparisons, indicating that untreated patients have high levels of anti-alpha-Gal antibodies throughout the course of infection.
[040] Amostras de soro individuais de dois pacientes chagásicos que receberam benznidazol foram coletadas 1 , 6, 12, 18, e 24 meses após o tratamento. O soro de dois outros pacientes ChP que não receberam tratamento foram coletados nos mesmos intervalos de tempo. Essas amostras foram testadas para anticorpos anti-alfa-Gal para acompanhar o curso da antigenicidade contra Q§ ( -Gal)540 nesses pacientes. Comparados com os dados obtidos a partir dos pacientes não tratados, as amostras de soro de pacientes que receberam benznidazol mostraram uma titulação de anticorpos anti-alfa-Gal menor (Figura 5A). Em um segundo teste, amostras de soro individuais coletadas em diferentes tempos de cinco outros pacientes chagásicos tratados foram testadas por ELISA contra Q§ ( -Gal)540, e também apresentaram níveis mais baixos de anti-alfa-Gal após tratamento com benznidazol (Figura 5B). Este padrão persistiu por longos períodos, como representado pelo paciente TP019, que foi acompanhado por 95 meses. [040] Individual serum samples from two chagasic patients receiving benznidazole were collected 1, 6, 12, 18, and 24 months after treatment. Serum from two other untreated ChP patients were collected at the same time intervals. These samples were tested for anti-alpha-Gal antibodies to follow the course of antigenicity against Q§ (-Gal) 540 in these patients. Compared with data from untreated patients, serum samples from patients receiving benznidazole showed lower anti-alpha-Gal antibody titration (Figure 5A). In a second test, individual serum samples collected at different times from five other chagasic treated patients were tested by ELISA against Q§ (-Gal) 540, and also had lower anti-alpha-Gal levels after benznidazole treatment (Figure 5B). This pattern persisted for long periods, as represented by patient TP019, who was followed for 95 months.
EXEMPLO 4 ■ ASSOCIAÇÃO DAS PARTÍCULAS VIRAIS A PROTEÍNAS RECOMBINANTES EXAMPLE 4 ■ ASSOCIATION OF VIRAL PARTICLES WITH RECOMBINANT PROTEINS
[041 ] Doença de Chagas (DC), causada pelo hemoflagelado protozoário parasito Trypanosoma cruzi, é uma das doenças mais endémicas e infecciosas na América Latina. Cerca de 15 milhões de pessoas atualmente estão infectados e milhares irão a óbito a cada ano devido a complicações clínicas importantes, particularmente a cardiomiopatia. Devido ao aumento da mobilidade da população global, DC tornou-se uma infecção emergente nos EUA e Europa, onde estíma-se que milhares de indivíduos estão infectados com o parasito. Recentemente, apenas Benznidazol está disponível comercialmente para o tratamento da doença. Este medicamento apresenta eficácia relativamente baixa na fase crónica da infecção, e apresenta acentuada toxicidade durante o tratamento. Além disso, não há nenhuma vacina humana para prevenir e/ou tratar DC, apesar de numerosos estudos de vacina experimental. Embora os trabalhos recentes tenham apresentado resultados muito promissores com proteína/peptídeo recombinantes, e vacinas de DNA, tendo como alvos alguns epitopos imunodominantes de células T CD8+ na superfície do parasito, determinar o quanto estes epitopos são imunodominantes entre diferentes isolados de T. cruzi ainda se faz necessário. Uma vacina ideal deve promover proteção universal contra parasitos isolados de diferentes regiões e genótipos. Isto é, de longe, o maior desafio no desenvolvimento de vacinas humanas contra parasitos e, talvez, a principal razão que, até o momento, não há uma única vacina em uso clínico para prevenção e/ou tratamento de infecções parasitárias humanas. Antígenos de glicano, por outro lado, permanecem muito mal explorados como alvos vacinais, apesar do fato que glicoconjugados (glycolipoproteins e glicolipídios) são as principais moléculas presentes na superfície de trypomastigotas de T. cruzi e contenham epitopos altamente imunogênicos, não comumente encontrados em seres humanos. A este respeito, por exemplo, níveis muito elevados de anticorpos líticos, protetores anti- galactosyl (Anticorpos anti- Gal) são elicidados contra epitopos deste trisacarídeo presentes em glicoproteínas do parasito, tanto na fase aguda como na crónica da doença de Chagas humana. Portanto, com estas informações e também por dados preliminares obtidos em nosso laboratório, formulamos a hipótese que o trisacarídeo terminal contendo Gal podem funcionar efetivamente como antígenos vacinais na doença de Chagas experimental e humana. Ao longo dos anos, vários grupos vêm tentando desenvolver uma vacina experimental, utilizando tanto parasitos atenuados quanto lisados do parasito, antígenos recombinantes ou purificados. Seus esforços podem ter sido constantemente prejudicados pela falta de modelos experimentais que mimetizem a resposta imune humana, principalmente relacionadas com a produção de anticorpos líticos. Outro obstáculo importante no desenvolvimento de uma vacina para o T. cruzi é a falta de adjuvantes potentes, que eficientemente melhorem a eficácia da resposta imune inata. [041] Chagas disease (CD), caused by the parasitic protozoan hemoflagellate Trypanosoma cruzi, is one of the most endemic and infectious diseases in Latin America. About 15 million people are currently infected and thousands will die each year due to major clinical complications, particularly cardiomyopathy. Due to increased mobility of the global population, DC has become an emerging infection in the US and Europe, where it is estimated that thousands of individuals are infected with the parasite. Recently, only Benznidazole is commercially available for the treatment of the disease. This medicine has relatively low efficacy in the chronic phase of infection, and has marked toxicity during treatment. In addition, there is no human vaccine to prevent and / or treat CD, despite numerous experimental vaccine studies. Although recent work has shown very promising results with recombinant protein / peptide and DNA vaccines targeting some immunodominant epitopes of CD8 + T cells on the parasite surface, determining how immunodominant these epitopes are among different T. cruzi isolates is necessary. An ideal vaccine should promote universal protection against parasites isolated from different regions and genotypes. This is by far the biggest challenge in the development of human parasite vaccines and perhaps the main reason that, so far, there is not a single vaccine in clinical use for prevention and / or treatment of human parasitic infections. Antigens of Glycans, on the other hand, remain very poorly exploited as vaccine targets, despite the fact that glycoconjugates (glycolipoproteins and glycolipids) are the major molecules present on the surface of T. cruzi trypomastigotes and contain highly immunogenic epitopes not commonly found in humans. In this regard, for example, very high levels of lytic antibodies, anti-galactosyl protectors (Anti-Gal Antibodies) are elicited against epitopes of this trisaccharide present on parasite glycoproteins, both in the acute and chronic phase of human Chagas disease. Therefore, with this information and also preliminary data obtained in our laboratory, we hypothesized that the terminal gal-containing trisaccharide can function effectively as vaccine antigens in experimental and human Chagas disease. Over the years, several groups have been trying to develop an experimental vaccine using both attenuated and lysed parasites of the parasite, either recombinant or purified antigens. Their efforts may have been constantly hampered by the lack of experimental models that mimic the human immune response, mainly related to the production of lytic antibodies. Another important obstacle in the development of a T. cruzi vaccine is the lack of potent adjuvants that efficiently improve the effectiveness of the innate immune response.
[042] Recentemente em nosso laboratório, utilizamos um modelo de camundongos Knock out para enzima 1 ,3-galactosyltransferase ( GalT-KO). Este modelo animal mimetiza de maneira muito próxima a resposta imune humoral humana contra o T. cruzi. Estes animais não expressam o epítopo Gal na superfície de suas células e tecidos, assim como macacos do velho mundo e humanos. Como Imunógeno, nós empregamos o trissacarídeo sintético, Gal 1 ,3Gal<f 1 ,4GlcNAc ( Gal) ligado ao virus QB-VLP, associados ou não a proteínas recombinantes como AdASP-2. Camundongos GalT-KO foram imunizados de acordo com o protocolo abaixo. Em contraste com os controles, os quais morreram dentro de 12 semanas, todos os camundongos imunizados com as partículas sobreviveram até 50 semanas, apresentando também baixa parasitemia e menor perda de peso (experimento de ponto de extremidade) seguidos por desafios letal parasitos (cepa Y), Figura 6. [042] Recently in our lab, we used a Knock out mouse model for enzyme 1,3-galactosyltransferase (GalT-KO). This animal model closely mimics the human humoral immune response against T. cruzi. These animals do not express the Gal epitope on the surface of their cells and tissues, just like old world monkeys and humans. As an immunogen, we employ the synthetic trisaccharide Gal 1, 3Gal <f 1,4,4GlcNAc (Gal) linked to the QB-VLP virus associated with or not to recombinant proteins like AdASP-2. GalT-KO mice were immunized according to the protocol below. In contrast to controls, which died within 12 weeks, all mice immunized with the particles survived up to 50 weeks, also presenting low parasitemia and lower weight loss (endpoint experiment) followed by lethal parasite challenges (strain Y ), Figure 6.

Claims

REIVINDICAÇÕES
1. Partícula virai Q-beta, cabracterizada por estar ligada a várias cópias do trissacarídeo sintético alfa-Gal (Gal 1 -3Gal 1 -4GlcNAcR).1. Q-beta viral particle characterized by being bound to several copies of the synthetic alpha-Gal trisaccharide (Gal 1 -3Gal 1 -4GlcNAcR).
2. Partícula virai Q-beta, de acordo com a reivindicação 1 , caracterizada por compreender de 180 a 600 cópias do trissacarídeo sintético alfa-Gal (Gal 1 -3Gal 1 -4GlcNAcR). Q-beta viral particle according to claim 1, comprising 180 to 600 copies of the synthetic alpha-Gal trisaccharide (Gal 1 -3Gal 1 -4GlcNAcR).
3. Partícula virai Q-beta, de acordo com a reivindicação 1 , caracterizada por estar associada ou não a proteínas recombinantes, preferencialmente a AdASP-2.  Q-beta viral particle according to Claim 1, characterized in that it is associated or not with recombinant proteins, preferably AdASP-2.
4. Método para o diagnóstico da Doença de Chagas, caracterizado por compreender as seguintes etapas:  4. Method for the diagnosis of Chagas disease, characterized by the following steps:
a) exposição de uma amostra a pelo menos uma das partícula virais Q-beta ligadas a várias cópias do trissacarídeo sintético alfa-Gal (Gal 1 -3Gal 1 -4GlcNAcR), estando tais partículas ligadas a um suporte sólido ou a um carreador;  (a) exposing a sample to at least one of the Q-beta viral particles bound to several copies of the synthetic alpha-Gal trisaccharide (Gal 1 -3Gal 1 -4GlcNAcR), such particles being bound to a solid support or a carrier;
b) adição de um anticorpo secundário ou uma proteína, que estejam conjugados a uma enzima ou a um marcador e que se liguem aos anticorpos da amostra da etapa (a);  b) adding a secondary antibody or protein which is conjugated to an enzyme or a marker and which binds to the antibodies of the sample from step (a);
c) detecção dos anticorpos específicos para T. cruzi na amostra citada na etapa (a), utilizando-se reagentes capazes de detectar a enzima ou o marcador citados na etapa (b). c) detecting T. cruzi specific antibodies in the sample cited in step (a) using reagents capable of detecting the enzyme or marker cited in step (b).
5. Método para diagnóstico da Doença de Chagas, de acordo com a reivindicação 4, etapa a_, caracterizado pelas amostras utilizadas serem selecionadas do grupo compreendendo sangue, soro, plasma e/ou outro fluído corporal. Method for the diagnosis of Chagas Disease according to claim 4, step a, characterized in that the samples used are selected from the group comprising blood, serum, plasma and / or other body fluid.
6. Método para diagnóstico da Doença de Chagas, de acordo com a reivindicação 4, etapa b_, caracterizado pelo anticorpo ser selecionado do grupo compreendendo IgG, IgM, IgA, IgE e/ou respectivas subclasses. Method for the diagnosis of Chagas disease according to claim 4, step b_, characterized in that the antibody is selected from the group comprising IgG, IgM, IgA, IgE and / or their subclasses.
7. Método para diagnóstico da Doença de Chagas, de acordo com a reivindicação 4, etapa b_, caracterizado pela proteína ser preferencialmente a Proteína A e/ou Proteína G.  Method for the diagnosis of Chagas disease according to claim 4, step b_, characterized in that the protein is preferably Protein A and / or Protein G.
8. Método para diagnóstico da Doença de Chagas, de acordo com a reivindicação 4, etapa b_, caracterizado pela enzima ser selecionada do grupo compreendendo peroxidase, fosfatase alcalina, beta-galactosidase, urease, xantina oxidase, glicose oxidase e penicilinase.  Method for the diagnosis of Chagas Disease according to claim 4, step b, characterized in that the enzyme is selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.
9. Método para o diagnóstico da Doença de Chagas, de acordo com a reivindicação 4, etapa b_, caracterizado pelo marcador ser selecionado do grupo compreendendo enzimas, radioisótopos, biotina, cromóforos, fluoróforos e quimioluminescentes.  Method for the diagnosis of Chagas disease according to claim 4, step b, characterized in that the marker is selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent.
10. Método para o diagnóstico da Doença de Chagas, de acordo com a reivindicação 4, etapa c_, caracterizado pela detecção do anticorpo secundário ou proteína especificamente ligados aos anticorpos anti- 7". cruzi ser feita utilizando reagentes selecionados do grupo compreendendo substratos cromógenos que sejam reconhecidos por alguma das enzimas da reivindicação 6 ou algum dos marcadores da reivindicação 7. Method for the diagnosis of Chagas Disease according to claim 4, step c, characterized in that the detection of the secondary antibody or protein specifically bound to the anti-7 ' antibodies is performed using reagents selected from the group comprising chromogenic substrates which are recognized by any of the enzymes of claim 6 or any of the markers of claim 7.
11. Kit para o diagnóstico da Doença de Chagas caracterizado por compreender:  11. Kit for the diagnosis of Chagas disease characterized by:
a) suporte sólido ou carreador contendo pelo menos uma das partículas virais Q-beta ligadas a várias cópias do trissacarídeo sintético alfa-Gal (Gal 1 -3Gal 1 -4GlcNAcR);  a) solid or carrier support containing at least one of the Q-beta viral particles bound to multiple copies of the alpha-Gal synthetic trisaccharide (Gal 1 -3Gal 1 -4GlcNAcR);
b) anticorpo secundário ou uma proteína, conjugados a uma enzima ou a um marcador;  b) secondary antibody or protein, conjugated to an enzyme or a marker;
c) reagente para detectar a enzima ou marcador. c) reagent for detecting the enzyme or label.
12. Kit para o diagnóstico da Doença de Chagas, de acordo com a reivindicação 11 , item a_, caracterizado pelo suporte sólido ser selecionado do grupo de materiais compreendendo nitrocelulose, nylon, látex, polipropileno e/ou poliestireno. Chagas disease diagnosis kit according to claim 11, item a, characterized in that the solid support is selected from the group of materials comprising nitrocellulose, nylon, latex, polypropylene and / or polystyrene.
13. Kit para o diagnóstico da Doença de Chagas, de acordo com a reivindicação 11 , itens b_ e c_, caracterizado pelas enzimas, marcadores e reagentes serem selecionados dos grupos descritos nas reivindicações 4 a 8.  Chagas disease diagnosis kit according to claim 11, items b and c, characterized in that the enzymes, markers and reagents are selected from the groups described in claims 4 to 8.
14. Uso das partículas virais definidas na reivindicação 1 , caracterizado por ser no diagnóstico e/ou em kits para diagnóstico da doença de Chagas.  Use of the viral particles as defined in claim 1, characterized in that they are for diagnosis and / or diagnostic kits for Chagas disease.
15. Vacina contra Doença de Chagas caracterizada por compreender as partículas virais conforme definidas na reivindicação 1 e excipientes farmaceuticamente aceitáveis.  Chagas disease vaccine comprising viral particles as defined in claim 1 and pharmaceutically acceptable excipients.
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