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Ehrlichia ruminantium is an obligate intracellular pathogen and, as such, cell mediated immunity plays a key role in the control of bacterial replication and subsequent protection against heartwater in ruminants. IFN-γ has been shown to be a potent inhibitor of E. ruminantium growth in endothelial cells in vitro. Thus, identification of antigens that preferentially activate T cells to proliferate and secrete IFN-γ needs to be done so that they can be evaluated as vaccine candidates. Previously, a cocktail of four open reading frames (ORFs) that induce 100% protection after needle challenge have been identified. However, only 20% protection was obtained after tick challenge in the field, when administered as a DNA vaccine in sheep. Because only limited protection was obtained during a field vaccine trial, our research focused on the identification of additional ORFs as a mean to improve the efficacy of this vaccine. Because secreted proteins are reported to be major targets in a specific immune response we hypothesize that they may be potential heartwater vaccine candidates. Five ORFs (Erum5000, Erum5010, Erum7760, Erum8060 and Erum8610) encoding secreted E. ruminantium proteins were selected from the Welgevonden stock genome sequence using bioinformatics tools. The ORFs were cloned into a pET 102/D-TOPO® vector. The corresponding recombinant (r) proteins were expressed in a bacterial expression system and the expression was confirmed by immunoblots using anti-His antibodies and sheep sera. Proteins were purified by immobilized metal ion affinity chromatography and resulted in a yield of 200 μg-2000 μg per 100 ml and 1L cultures respectively. Four of the five recombinant proteins (rErum5000, rErum7760, rErum8060 and rErum8610) could be expressed. Recombinant proteins were assayed to determine whether they induce recall cellular immune responses in vitro. The peripheral blood mononuclear cells used in the assays were obtained from a naïve and four heartwater immune sheep. Significant proliferative responses (p ≤ 0.01) were evident for 3/4 recombinant proteins (rErum5000, rErum8060 and rErum8610). IFN-γ production was determined using an ELISPOT assay and 3/4 recombinant proteins (rErum5000, rErum8060 and rErum8610) induced IFN-γ production. Each recombinant protein had its own optimum concentration for inducing immune responses and the responses differed between animals. In addition, real-time PCR was used to measure IFN-γ and IL-4 gene expression by antigen stimulated immune PBMC. The real-time PCR results correlated with the ELISPOT assay results. Based on the results obtained, it can be concluded that a Th1 type immune response was elicited. Thus these proteins that induced proliferation and IFN-γ production may be important in protection against heartwater and will be tested in future vaccine studies. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / Unrestricted

Professional antigen presenting cells (pAPCs) process and present antigens on their cell surface in association with MHC class I molecules through two general pathways: direct or cross-presentation. The process of antigen presentation by pAPCs to naïve T cells resulting in their proliferation and differentiation into activated cytotoxic lymphocytes (CTLs) is called T cell priming. In these studies, we examine the cross-presentation of antigens from two non-replicating viruses: inactivated Lymphocytic Choriomeningitis virus (LCMV), and recombinant baculovirus encoding the LCMV nucleoprotein (NP). Since effective activation of pAPCs is essential for efficient priming of CD8+ T cells and CTL activation, and because infection with inactivated viruses generally induces an extremely poor level of CTL activation, we examined the activation state of pAPCs by measuring their cytokine profiles following infection to help further delineate their involvement in the CTL response to inactivated viruses. Our results indicate a pro-inflammatory cytokine mRNA upregulation in pAPCs in response to the inactivated virus, similar to the cytokine profiles subsequent to live LCMV infection, but to a lesser extent. In these studies, we also examined CTL activation following infection with inactivated LCMV and bAc-NP. We have demonstrated that the presentation of antigens from inactivated LCMV and bAC-NP results in a low level of CTL activation in vivo, though there is an undetectable level of CTL activation in vitro, in comparison to activation following infection with the live virus. Ultimately, the characterization of the cytokine profiles of pAPCs and the CD8+ T cell profiles induced in response to inactivated LCMV or the baculovirus derived NP may lead to a better understanding of how cross-presentation of these viral antigens may occur. This information may be applied to enhance the process of pAPC activation and T cell priming, for the induction of more effective cellular immune responses and the generation of stronger protective immunity. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-09-02 15:30:13.883

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2009 / Background: RTS,S candidate malaria vaccine has been shown to be highly immunogenic in children and infants, but the protective immune mechanisms still remain to be clearly elucidated. It is believed that RTS,S elicits a strong neutralizing humoral immune response directed against surface-exposed sporozoite proteins and cell mediated immune (CMI) responses characterized by predominantly CD4+ Th1 cells. The objective of this study was to investigate humoral and cell-mediated immune responses to the RTS,S/AS02D malaria vaccine and its association with protection against infection and disease by P. falciparum. Methodology and Principal Findings: This secondary data analysis from data of a phase I/IIb randomized, double-blind, controlled trial, included 154 healthy infants living in rural Mozambique, previously immunized with RTS,S/AS02D candidate malaria vaccine or the control Engerix-B™ vaccine. Antibodies against circumsporozoite protein (CSP) and hepatitis-B surface antigen (HBsAg) were measured with a standard ELISA. Fresh blood intracellular staining assay was performed to evaluate the expression of IL-2 and IFN-γ by CD4+ and CD8+ cells in response to in vitro stimulation of specific peptides. Data was evaluated for association with the risk of malaria detected by both active and passive case detection of infection over a period of 6 months post dose 3. Anti-HBs antibody geometric mean titers declined from 10,082 mIU/mL one month post Dose 3 to 2,751 mIU/mL at 12 months post Dose 3 in the RTS,S/AS02D group; anti-HBs v geometric mean titers were 392.4 mIU/mL and 263.9 mIU/mL, respectively in the Engerix- BTM group. Anti-CSP antibody geometric mean titers declined from 199.9 EU/mL one month post Dose 3 to 7.3 EU/mL at 12 months post Dose 3 in the RTS,S/AS02D group. Median stimulation indices of HBs-specific IL-2 and IFN-γ producing CD8+ T cells was higher in the RTS,S/AS02D group than in control group (Wilcoxon rank sum p-values for IFN-γ = 0.015, for IL-2 = 0.030) at 10.5 weeks post immunization. Median stimulation indices of anti-CSP specific IFN-γ producing CD8+ T cells at the same time point was 1.13 (IQR: 0.79 - 1.67; p=0.029). For specific IL-2-producing CD4+ T cells, the median SI was 1.14 (IQR: 0.74 – 1.60, p=0.043) at 10.5 weeks post dose three. The reduction in hazards of malaria infection were 18.3 % (95% CI: -267.9 – 81.8, p=0.793) and -12.0 % (95% CI: -295 – 68.2, p=0.86) for specific IL-2 CD4+ stimulation indices; For specific CD8+ IFN-γ stimulation indices the hazards were -103.6% (95% CI: -690.9 – 47.6; p=0.305) and 48.8% (95% CI: -97.0 – 86.7; p=0.33) at four and 10.5 weeks post immunization respectively. Conclusion: The RTS,S/AS02D vaccine was immunogenic and has elicited detectable levels of CSP specific cell mediated responses. No evidence of association was found between the antibodies anti-CSP and specific cell mediated responses and the risk of malaria.

Background: The prevalence of immunological disorders such as type 1 diabetes (T1D) is increasingly common amongst children, adolescents and young adults. There is also an increase in psychosomatic symptoms (depression, insomnia, anxiety, headaches and fatigue etc.) as well as a decrease in physical activity amongst young people, affecting the well-being and overall health of our younger population. It is therefore important to study the effects of psychological and physical stressors on the immune system, to evaluate their impact on juvenile health. Aim: This thesis explores the impact of psychological and physical stressors on the cellular immune system with special focus on diabetes-related immunity in a young population, using an interdisciplinary approach. Method: When exploring the impact of psychological and physical stressors such as psychological stress due to exposure to psychological stressful experiences or degree of physical activity/training on the cellular immune system in children, adolescents and young women, peripheral blood mononuclear cells (PBMC) were stimulated with antigens (tetanus toxoid (TT) and β-lactoglobulin (βLG)) as well as diabetes-related autoantigens (insulin, heat shock protein 60 (HSP60), tyrosine phosphatase-2 (IA-2) and glutamic acid decarboxylase 65 (GAD65)) and secreted cytokines and chemokines were measured by multiplex fluorochrome technique (Luminex). Populations of Thelper (Th) cells (CD4+), T-cytotoxic (Tc) cells (CD8+), B cells (CD19+), Natural Killer (NK) cells (CD56+CD16+) as well as regulatory T (Treg) cells (CD4+CD25+FoxP3+CD127-), and their expression of CD39 and CD45RA were studied by flow cytometry. Diabetes-related parameters (glucose, C-peptide,proinsulin, pancreatic polypeptide and peptide YY) were measured to studyβ-cell activity and appetite regulation and cortisol was used as a biological marker for psychological and physical stress. Results: Children in families exposed to psychological stress showed an imbalanced cellular immune response as well as an increased immune response towards diabetes-related autoantigens. Also, previous exposure to psychological stress as well as current exposure to psychological stress in young women showed an increased immune response towards diabetes-related autoantigens. Further, previous exposure to psychological stress in young women showed increased numbers of circulating CD56+CD16+ NK cells as wellas decreased numbers of circulating CD4+CD25+FoxP3+CD127- Treg cells. High physical activity in children showed decreased spontaneous immune response as well as a decreased immune response towards diabetes-related autoantigens, while low physical activity in children showed an increased immune response towards diabetes-related autoantigens. Further, endurance training in adolescents, especially in adolescent males and young adolescents, showed an increased immune response towards the diabetes-related autoantigen IA-2. Conclusion: It is evident that psychological and physical stressors such as exposure to psychological stress and degree of physical activity/training impact the cellular immune system. Experiences associated with psychological stress seem to have a negative effect on the cellular immune system in a young population, causing an imbalance in the immune system that could possibly induce diabetes-related immunity. High physical activity in children seems to have a protective effect against diabetes-related immunity. In contrast, low physical activity in children and endurance training in adolescents seems to induce diabetes-related immunity. It is very likely that psychological stressful experiences, low physical activity and intense training such as endurance training all play important roles in the immunological process leading to the development of type 1 diabetes.

A malária continua sendo um dos maiores problemas de saúde pública no mundo e apesar dos muitos esforços no combate a doença, as estratégias disponíveis hoje ainda não foram capazes de controlar com eficiência sua disseminação global. Por isso o desenvolvimento de uma vacina é de extrema importância. Devido a complexidade do ciclo do parasita, consideramos que uma formulação vacinal eficaz para a indução de resposta imune protetora contra o P. vivax deverá conter regiões de antígenos de vários estágios do parasita. Dentre os vários antígenos da fase sanguínea do plasmódio identificados, o Antígeno 1 de Membrana Apical (AMA1), a porção C-terminal da Proteína 1 de Superfície do Merozoíto (MSP119) e as proteínas da família MSP3 têm se mostrado promissores. Portanto, o objetivo deste estudo foi caracterizar a resposta imune celular induzida em camundongos pela imunização experimental com antígenos de P. vivax candidatos a vacina, tendo como foco principal a combinação das proteínas AMA1, MSP119 e MSP3β. Inicialmente, as imunizações experimentais com a combinação dos 3 antígenos foram realizadas na presença de Adjuvante Incompleto de Freund (AIF) em camundongos C57BL/6. Os camundongos imunizados não apresentaram respostas significativas com proliferação e secreção de IFN-γ por linfócitos T CD4+ e CD8+ após estimulação in vitro com as proteínas recombinantes ou peptídeos sintéticos baseados na proteína AMA1. Por outro lado, o adjuvante Poly (I:C) mostrou-se mais eficiente na indução de resposta proliferativa por linfócitos T CD4+, quando testado com a proteína MSP119-PADRE. Assim, selecionamos este adjuvante para dar continuidade às imunizações experimentais com a combinação de antígenos. Por ELISPOT, foi possível demonstrar que a co-administração dos 3 antígenos foi capaz de estimular células produtoras de IFN-γ em resposta à todos os antígenos testados. Também foi nosso objetivo avaliar a produção de IFN-γ após imunização de linhagens distintas de camundongos com uma proteína quimérica recentemente produzida em nosso laboratório (AMA1-MSP119). Em C57BL/6, a produção de IFN-γ foi significativamente maior no grupo imunizado com a proteína de fusão, quando comparado com o grupo que recebeu a co-adminstração dos dois antígenos, mostrando que a utilização de proteínas fusionadas pode solucionar possíveis problemas de competição antigênica. Em conjunto, nossos dados demonstram que a utilização de múltiplos antígenos fusionados, ou co-administrados, pode ser uma estratégia promissora de vacinação contra a malária. / Malaria remains as a major public health problem worldwide. In spite of the efforts for prevention, the strategies available today have not succeeded to control its global distribution. Therefore, the development of a vaccine is extremely important. Due to the complexity of the parasite cycle, we consider that an effective vaccine formulation to induce protective immune response against P. vivax should contain regions of antigens from several stages of the parasite. Among the different blood stage antigens of Plasmodium identified, the Apical Membrane Antigen 1 (AMA1), the C-terminal portion of the Merozoite Surface Protein 1 (MSP119) and MSP3 family proteins have shown promising results. Therefore, the aim of this study was to characterize the cellular immune response induced in mice by experimental immunization with antigens from P. vivax vaccine candidates, focusing mainly on the combination of proteins AMA1, MSP119 and MSP3β. Initially, the experimental immunizations with the combination of the three antigens were performed in the presence of Incomplete Freund\'s Adjuvant (IFA) in C57BL/6 mice. Immunized mice displayed no significant proliferative responses and secretion of IFN-γ by CD4+ and CD8+ T lymphocytes after in vitro stimulation with recombinant proteins or synthetic peptides based on the AMA1 protein. In contrast, Poly (I:C) adjuvant was more efficient inducing CD4+ T lymphocytes proliferative response to the MSP119-PADRE protein. Thus, we selected this adjuvant to continue the experimental immunizations with the different antigen combinations. The ELISPOT results demonstrated that co-administration of the 3 antigens stimulated IFN-γ producing cells in response to all antigens tested. We also assessed the production of IFN-γ after immunization of distinct mouse strains with a chimeric protein produced recently in our laboratory (AMA1-MSP119). In C57BL/6, IFN-γ production was significantly higher when we used spleen cells from mice immunized with the fusion protein, when compared with cells from mice that received the co-administration of two antigens, showing that the use of fused proteins can solve possible antigenic competition problems. Together, our data demonstrate that the use of multiple fused or co-administered antigens may be a promising strategy for malaria vaccination.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-04T16:10:12Z No. of bitstreams: 1 Luana Leandro Gois Perfil... 2015.pdf: 11133579 bytes, checksum: 84c1592269b916a72d1e76ba863403c7 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-04T16:10:24Z (GMT) No. of bitstreams: 1 Luana Leandro Gois Perfil... 2015.pdf: 11133579 bytes, checksum: 84c1592269b916a72d1e76ba863403c7 (MD5) / Made available in DSpace on 2016-02-04T16:10:24Z (GMT). No. of bitstreams: 1 Luana Leandro Gois Perfil... 2015.pdf: 11133579 bytes, checksum: 84c1592269b916a72d1e76ba863403c7 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A infecção pelo HIV promove a redução do número de linfócitos T CD4+ e, consequentemente, o surgimento de doenças oportunistas. A leishmaniose visceral e a tuberculose são comumente reconhecidas como doenças oportunistas importantes e associadas ao óbito de indivíduos infectados por HIV. Ambos os patógenos, Leishmania e Mycobacterium tuberculosis (Mtb) infectam cronicamente macrófagos. A imunidade protetora associada a estas infecções envolve linfócitos Th1 produtores de IFN-g. O prejuízo na resposta imune celular causado pelo HIV perturba a resposta imune contra estes patógenos. Não são bem determinadas quais alterações imunológicas causadas pelo HIV promovem o prejuízo na resposta imune específica contra a Leishmania spp. e Mtb, induzindo o desenvolvimento de formas atípicas e graves destas infecções. Deste modo, esta tese teve como objetivo descrever o perfil da resposta imune celular aos antígenos de Leishmania spp. ou Mtb em pacientes infectados com HIV. Para tal., foram recrutados pacientes infectados por HIV e com diagnóstico de leishmaniose (HIV/LV) e tuberculose (HIV/TB). Indivíduos não infectados por HIV e diagnóstico de leishmaniose (LV) ou tuberculose (TB) forma incluídos como controles. Foram avaliadas a linfoproliferação e a frequência das subpopulações de memória dos linfócitos T CD4+ em resposta aos antígenos solúveis de Leishmania spp. (SLA). Igualmente, foram avaliadas a linfoproliferação, a frequência das subpopulações de memória dos linfócitos T CD4+ e CD8+, o perfil de funcional de linfócitos T CD4+ e CD8+ produtores de citocinas e a atividade citotóxica de linfócitos T CD8+ e células NK em resposta ao purificado protéico derivado (PPD) do M. bovis. Duas revisões sistemáticas da literatura que abordam a associação entre estas infecções e a síndrome inflamatória de reconstituição imune em indivíduos infectados por HIV após terapia antiretroviral foram realizadas. Os linfócitos T CD4+ e CD8+ dos pacientes HIV-LV não apresentavam resposta proliferativa ao SLA e houve redução na frequência de subpopulações de linfócitos T CD4+, a qual foi restaurada após o tratamento para leishmaniose visceral. Nos pacientes HIV-TB foi igualmente observada ausência de resposta proliferativa de linfócitos T CD4+ e CD8+ e de células produtoras de IFN-g em resposta ao PPD. Além disso, o HIV promoveu a redução da atividade degranulativa de células NK, o que contribui para o descontrole da infecção e desenvolvimento de TB ativa. Nos pacientes HIV-TB, HAART foi capaz de induzir uma recuperação parcial de células específicas produtoras de IFN-g, bem como da proliferação em resposta ao PPD. Em conjunto, os resultados desta tese sugerem que a infecção pelo HIV induz alterações na resposta celular de memória central e efetora contra patógenos intracelulares oportunistas. Essas alterações são parcialmente restauradas no curso da HAART. / The HIV-infection promotes reduced number of CD4+ T-lymphocytes and manifestation of opportunistic diseases. Visceral leishmaniasis and tuberculosis are commonly known as main opportunistic infections and are associated with mortality in HIV-infected individuals. Both pathogens, Leishmania and Mycobacterium tuberculosis (Mtb), infect macrophages. The protect immune response involve T-lymphocytes help 1 (Th1) and producing of IFN-g. The impairment of cellular immune response caused by HIV disrupts the immune response against these pathogens. It is unclear which immunological alterations caused by HIV infection promote the damage in specific cellular immune response against Leishmania and Mtb and induces the development of atypical and severe forms. Thus, this thesis aimed to describe the profile of the cellular immune response to Leishmania antigens or Mtb in HIV infected patients. To this end, were recruited HIV infected patients with visceral leishmaniasis (HIV/VL) and HIV infected patients with active tuberculosis (HIV/TB). Moreover, HIV uninfected individuals with VL or TB were also included as controls. Lymphoproliferation and frequency of memory CD4+ T-lymphocyte subsets in response to soluble Leishmania antigen (SLA) were evaluated. Also were evaluated lymphoproliferation, frequency of memory CD4+ and CD8+ T-lymphocyte subsets, functional profile of cytokines producing CD4+ and CD8+ T-lymphocytes and cytotoxic activity of CD8+ Tlymphocytes and NK cells in response to purified protein derivative (PPD) of M. bovis. Two systematic reviews of the literature concerning the association between leishmaniasis and tuberculosis with the inflammatory immune reconstitution syndrome (IRIS) in HIV-infected individuals after antiretroviral therapy were done. The absence of proliferative response to SLA of CD4+ and CD8+ T-lymphocytes and the reduced frequency of memory CD4+ T-lymphocyte subsets were observed in HIV/VL patients. The frequency of memory CD4+ T-lymphocyte subset was restored after treatment for visceral leishmaniasis. In HIV/TB patients was also observed absence of proliferative response to PPD of CD4+ and CD8+ T-lymphocytes and of PPD-specific IFN-g producing cells. In addition, HIV infection promotes the reduction in degranulative activity of NK cells which contributes to the survival of Mtb into macrophages and development of active TB. In HIV/TB patients, HAART was able to induce a partial recovery of PPD-specific IFN-g producing cells and of lymphoproliferation in response to PPD. Taken together, the results suggest HIV infection induces changes in cellular immune response of central and effector memory against opportunistic intracellular pathogens. These alterations are partially restored in the after HAART. Keywords: HIV,

Orientadora: Mary Marcondes / Banca: Raimundo Souza Lopes / Banca: Vera Lúcia Fonseca de Camargo Neves / Resumo: A leishmaniose visceral pode ser incluída como uma das causas de miopatia inflamatória em cães, entretanto, pouco se sabe sobre a patogênese da doença no sistema muscular, sendo incriminada muitas vezes apenas à natureza catabólica da enfermidade. O objetivo deste estudo foi avaliar, por meio de imunoistoquímica, a presença de formas amastigotas de Leishmania sp, linfócitos T (CD3+), macrófagos e IgG nos músculos tríceps braquial, extensor carpo radial, bíceps femoral e gastrocnêmio de 23 cães naturalmente acometidos por leishmaniose visceral. Dentre os 92 músculos avaliados,11 (12%) apresentaram marcação antigênica para formas amastigotas de Leishmania sp, 35 (38,1%) para linfócitos T (CD3+), 29 (31,5%) para macrófagos e 14 (12%) para IgG. Os resultados obtidos permitiram concluir que em cães com leishmaniose visceral apresentam imunomarcação para formas amastigotas de Leishmania sp., linfócitos T CD3+, macrófagos e IgG, sugerindo a participação direta do parasito e de uma resposta imune celular e humoral na fisiopatogenia da lesão muscular. / Abstract: Visceral leishmaniasis may be included as a cause of inflammatory myophathy in dogs, however, little is known about the pathogenesis of the disease in the muscular system, which is frequently associated with the catabolic nature of the illness. The purpose of this study was investigate, through immunohistochemistry, the presence of amastigote forms of Leishmania sp, T lymphocytes (CD3+), macrophages and IgG in the muscle triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius of 23 dogs with visceral leishmaniasis. Among 92 evaluated muscles, 11 (12%) presented antigenic marking for amastigote forms of Leishmania sp., 35 (38,1%) for T lymphocites (CD3+), 29 (31,5%) for macrophages and 14 (12%) for IgG. The results of the present experiment led to the conclusion that in dogs with visceral leishmaniasis there may be a straight participation of the parasite and of cellular and humoral immune response in the ethiopatogeny of the muscular injury. / Mestre

A malária continua sendo um dos maiores problemas de saúde pública no mundo e apesar dos muitos esforços no combate a doença, as estratégias disponíveis hoje ainda não foram capazes de controlar com eficiência sua disseminação global. Por isso o desenvolvimento de uma vacina é de extrema importância. Devido a complexidade do ciclo do parasita, consideramos que uma formulação vacinal eficaz para a indução de resposta imune protetora contra o P. vivax deverá conter regiões de antígenos de vários estágios do parasita. Dentre os vários antígenos da fase sanguínea do plasmódio identificados, o Antígeno 1 de Membrana Apical (AMA1), a porção C-terminal da Proteína 1 de Superfície do Merozoíto (MSP119) e as proteínas da família MSP3 têm se mostrado promissores. Portanto, o objetivo deste estudo foi caracterizar a resposta imune celular induzida em camundongos pela imunização experimental com antígenos de P. vivax candidatos a vacina, tendo como foco principal a combinação das proteínas AMA1, MSP119 e MSP3β. Inicialmente, as imunizações experimentais com a combinação dos 3 antígenos foram realizadas na presença de Adjuvante Incompleto de Freund (AIF) em camundongos C57BL/6. Os camundongos imunizados não apresentaram respostas significativas com proliferação e secreção de IFN-γ por linfócitos T CD4+ e CD8+ após estimulação in vitro com as proteínas recombinantes ou peptídeos sintéticos baseados na proteína AMA1. Por outro lado, o adjuvante Poly (I:C) mostrou-se mais eficiente na indução de resposta proliferativa por linfócitos T CD4+, quando testado com a proteína MSP119-PADRE. Assim, selecionamos este adjuvante para dar continuidade às imunizações experimentais com a combinação de antígenos. Por ELISPOT, foi possível demonstrar que a co-administração dos 3 antígenos foi capaz de estimular células produtoras de IFN-γ em resposta à todos os antígenos testados. Também foi nosso objetivo avaliar a produção de IFN-γ após imunização de linhagens distintas de camundongos com uma proteína quimérica recentemente produzida em nosso laboratório (AMA1-MSP119). Em C57BL/6, a produção de IFN-γ foi significativamente maior no grupo imunizado com a proteína de fusão, quando comparado com o grupo que recebeu a co-adminstração dos dois antígenos, mostrando que a utilização de proteínas fusionadas pode solucionar possíveis problemas de competição antigênica. Em conjunto, nossos dados demonstram que a utilização de múltiplos antígenos fusionados, ou co-administrados, pode ser uma estratégia promissora de vacinação contra a malária. / Malaria remains as a major public health problem worldwide. In spite of the efforts for prevention, the strategies available today have not succeeded to control its global distribution. Therefore, the development of a vaccine is extremely important. Due to the complexity of the parasite cycle, we consider that an effective vaccine formulation to induce protective immune response against P. vivax should contain regions of antigens from several stages of the parasite. Among the different blood stage antigens of Plasmodium identified, the Apical Membrane Antigen 1 (AMA1), the C-terminal portion of the Merozoite Surface Protein 1 (MSP119) and MSP3 family proteins have shown promising results. Therefore, the aim of this study was to characterize the cellular immune response induced in mice by experimental immunization with antigens from P. vivax vaccine candidates, focusing mainly on the combination of proteins AMA1, MSP119 and MSP3β. Initially, the experimental immunizations with the combination of the three antigens were performed in the presence of Incomplete Freund\'s Adjuvant (IFA) in C57BL/6 mice. Immunized mice displayed no significant proliferative responses and secretion of IFN-γ by CD4+ and CD8+ T lymphocytes after in vitro stimulation with recombinant proteins or synthetic peptides based on the AMA1 protein. In contrast, Poly (I:C) adjuvant was more efficient inducing CD4+ T lymphocytes proliferative response to the MSP119-PADRE protein. Thus, we selected this adjuvant to continue the experimental immunizations with the different antigen combinations. The ELISPOT results demonstrated that co-administration of the 3 antigens stimulated IFN-γ producing cells in response to all antigens tested. We also assessed the production of IFN-γ after immunization of distinct mouse strains with a chimeric protein produced recently in our laboratory (AMA1-MSP119). In C57BL/6, IFN-γ production was significantly higher when we used spleen cells from mice immunized with the fusion protein, when compared with cells from mice that received the co-administration of two antigens, showing that the use of fused proteins can solve possible antigenic competition problems. Together, our data demonstrate that the use of multiple fused or co-administered antigens may be a promising strategy for malaria vaccination.