Induction of immunogenicity by live attenuated Leishmania donovani centrin deleted parasites in dogs

AbstractZoonotic visceral leishmaniasis, caused by the intracellular protozoan parasite Leishmania infantum, is a neglected tropical disease that is often fatal when untreated. Dogs are considered the main reservoir of L. infantum in zoonotic VL as the presence of infected dogs may increase the risk for human infection. Canine visceral leishmaniasis (CVL) is a major veterinary and public health problem in Southern Europe, Middle East and South America. Control of animal reservoirs relies on elimination of seropositive dogs in endemic areas. However, treatment of infected dogs is not considered a favorable approach as this can lead to emergence of drug resistance since the same drugs are used to treat human infections. Therefore, vaccination against CVL remains the best alternative in control of the animal reservoirs. In this study, we present data on the immunogenicity profile of a live attenuated parasite LdCen−/− in a canine infection model and compared it to that of Leishmune®, a commercially available recombinant vaccine. The immunogenicity of the LdCen−/− parasites was evaluated by antibody secretion, production of intracytoplasmic and secreted cytokines, activation and proliferation of T cells. Vaccination with LdCen−/− resulted in high immunogenicity as revealed by the higher IgGTotal, IgG1, and IgG2 production and higher lymphoproliferative response. Further, LdCen−/− vaccinated dogs showed higher frequencies of activated CD4+ and CD8+ T cells, IFN-γ production by CD8+ T cells, increased secretion of TNF-α and IL-12/IL-23p40 and decreased secretion of IL-4. These results contribute to the understanding of immunogenicity elicited by live attenuated L. donovani parasites and, consequently, to the development of effective vaccines against visceral leishmaniasis

Immunoregulatory mechanisms in Chagas disease: modulation of apoptosis in T-cell mediated immune responses

Submitted by Nuzia Santos ([email protected]) on 2017-07-17T17:53:45Z No. of bitstreams: 1 Chaves_AnaThereza_Immunoregulatory mechanisms_IRR_2016.pdf: 12736177 bytes, checksum: 7182dae7e3675c77254aa3dd4157a0a9 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2017-07-17T18:03:09Z (GMT) No. of bitstreams: 1 Chaves_AnaThereza_Immunoregulatory mechanisms_IRR_2016.pdf: 12736177 bytes, checksum: 7182dae7e3675c77254aa3dd4157a0a9 (MD5)Made available in DSpace on 2017-07-17T18:03:09Z (GMT). No. of bitstreams: 1 Chaves_AnaThereza_Immunoregulatory mechanisms_IRR_2016.pdf: 12736177 bytes, checksum: 7182dae7e3675c77254aa3dd4157a0a9 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Morfologia. Laboratório de Biologia das Interações Celulares. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Faculdade de Medicina Programa de Pós graduação em Medicina Tropical e Infectologia. Belo Horizonte, MG, Brazil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Biomarcadores de Diagnóstico e Monitoração. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Morfologia. Laboratório de Biologia das Interações Celulares. Belo Horizonte, MG, Brazil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, BrazilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Laboratório de Imunologia e Genômica de Parasitos. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Fisiologia e Biofísica. Belo Horizonte, MG, Brazil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, BrazilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Laboratório de Imunologia e Genômica de Parasitos. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Faculdade de Medicina Programa de Pós graduação em Medicina Tropical e Infectologia. Belo Horizonte, MG, Brazil.Laboratório de Imunologia Celular e Molecular, Centro de Pesquisas René Rachou, Fiocruz, Belo Horizonte, Brazil/Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais. Belo Horizonte, MG, Brazil/Universidade Federal de Ouro Preto. Ouro Preto, MG, Brazil.BACKGROUND: Chronic Chagas disease presents different clinical manifestations ranging from asymptomatic (namely indeterminate) to severe cardiac and/or digestive. Previous results have shown that the immune response plays an important role, although no all mechanisms are understood. Immunoregulatory mechanisms such as apoptosis are important for the control of Chagas disease, possibly affecting the morbidity in chronic clinical forms. Apoptosis has been suggested to be an important mechanism of cellular response during T. cruzi infection. We aimed to further understand the putative role of apoptosis in Chagas disease and its relation to the clinical forms of the disease. METHODS: Apoptosis of lymphocytes, under antigenic stimuli (soluble T. cruzi antigens - TcAg) where compared to that of non-stimulated cells. Apoptosis was evaluated using the expression of annexin and caspase 3(+) by T cells and the percentage of cells positive evaluated by flow cytometry. In addition activation and T cell markers were used for the identification of TCD4(+) and TCD8(+) subpopulations. The presence of intracellular and plasma cytokines were also evaluated. Analysis of the activation status of the peripheral blood cells showed that patients with Chagas disease presented higher levels of activation determined by the expression of activation markers, after TcAg stimulation. PCR array were used to evaluate the contribution of this mechanism in specific cell populations from patients with different clinical forms of human Chagas disease. RESULTS: Our results showed a reduced proliferative response associated a high expression of T CD4(+)CD62L(-) cells in CARD patients when compared with IND group and NI individuals. We also observed that both groups of patients presented a significant increase of CD4(+) and CD8(+) T cell subsets in undergoing apoptosis after in vitro stimulation with T. cruzi antigens. In CARD patients, both CD4(+) and CD8(+) T cells expressing TNF-α were highly susceptible to undergo apoptosis after in vitro stimulation. Interestingly, the in vitro TcAg stimulation increased considerably the expression of cell death TNF/TNFR superfamily and Caspase family receptors genes in CARD patients. CONCLUSIONS: Taken together, our results suggest that apoptosis may be an important mechanism for the control of morbidity in T. cruzi infection by modulating the expression of apoptosis genes, the cytokine environment and/or killing of effector cells

Profile of Central and Effector Memory T Cells in the Progression of Chronic Human Chagas Disease

Chagas disease is a parasitic infection caused by protozoan Trypanosoma cruzi that affects approximately 11 million people in Latin America. The involvement of the host's immune response on the development of severe forms of Chagas disease has not been fully elucidated. Studies on the immune response against T. cruzi infection show that the immunoregulatory mechanisms are necessary to prevent the deleterious effect of excessive immune response stimulation and consequently the fatal outcome of the disease. A recall response against parasite antigens observed in in vitro peripheral blood cell culture clearly demonstrates that memory response is generated during infection. Memory T cells are heterogeneous and differ in both the ability to migrate and exert their effector function. This heterogeneity is reflected in the definition of central (TCM) and effector memory (TEM) T cells. Our results suggest that a balance between regulatory and effectors T cells may be important for the progression and development of the disease. Furthermore, the high percentage of central memory CD4+ T cells in indeterminate patients after stimulation suggests that these cells may modulate host's inflammatory response by controlling cell migration to tissues and their effector role during chronic phase of the disease

Deletion of MIF gene from live attenuated LdCen −/− parasites enhances protective CD4+ T cell immunity

Abstract Vaccination with live attenuated Leishmania parasites such as centrin deleted Leishmania donovani (LdCen −/− ) against visceral leishmaniasis has been reported extensively. The protection induced by LdCen −/− parasites was mediated by both CD4+ and CD8+ T cells. While the host immune mediators of protection are known, parasite determinants that affect the CD4+ and CD8+ T cell populations remain unknown. Parasite encoded inflammatory cytokine MIF has been shown to modulate the T cell differentiation characteristics by altering the inflammation induced apoptosis during contraction phase in experimental infections with Leishmania or Plasmodium. Neutralization of parasite encoded MIF either by antibodies or gene deletion conferred protection in Plasmodium and Leishmania studies. We investigated if the immunogenicity and protection induced by LdCen −/− parasites is affected by deleting MIF genes from this vaccine strain. Our results showed that LdCen −/− MIF −/− immunized group presented higher percentage of CD4+ and CD8+ central memory T cells, increased CD8+ T cell proliferation after challenge compared to LdCen −/− immunization. LdCen −/− MIF −/− immunized group presented elevated production of IFN-γ+ and TNF-α+ CD4+ T cells concomitant with a reduced parasite load in spleen and liver compared to LdCen −/− group following challenge with L. infantum. Our results demonstrate the role of parasite induced factors involved in protection and long-term immunity of vaccines against VL

Aspects of the ecology and behaviour of phlebotomines in endemic area for visceral leishmaniasis in State of Minas Gerais

Submitted by Nuzia Santos ([email protected]) on 2012-12-11T16:05:42Z No. of bitstreams: 1 5. Aspectos da ecologia e do comportamento de flebotomíneos em.pdf: 39803 bytes, checksum: 56419ee60fd6f80a21af5ff9ec964244 (MD5)Made available in DSpace on 2012-12-11T16:05:42Z (GMT). No. of bitstreams: 1 5. Aspectos da ecologia e do comportamento de flebotomíneos em.pdf: 39803 bytes, checksum: 56419ee60fd6f80a21af5ff9ec964244 (MD5) Previous issue date: 2005Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Belo Horizonte, MG, Brasil.Fundação Nacional de Saúde. Belo Horizonte, MG, BrasilFaculdade de Medicina do Triângulo Mineiro. Uberaba, MG, BrasilFundação Oswalo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilO comportamento e hábitos alimentares de algumas espécies da flebotomíneos têm sido útil na compreensão da epidemiologia das leishmanioses. No município de Porteirinha (MG), foram realizadas capturas mensais sistematizadas utilizando-se 28 armadilhas luminosas tipo CDC, durante o período de janeiro a dezembro de 2002. Foram capturadas14 espécies de flebotomíneos, totalizando 1.408 exemplares. De acordo com o ambiente, os resultados obtidos mostraram que o peridomicílio apresentou a maior (53,3%) porcentagem dos espécimens encontrados na região, embora parte (46,7%) da fauna também tenha sido encontrada no intradomicílio. O repasto sanguíneo de 38 fêmeas de Lutzomyia longipalpis, provenientes do campo, foi identificado através da reação de precipitina. Os resultados indicam que Lutzomyia longipalpis foi a espécie predominante (65,1%), mostrando-se oportunista, podendo sugar uma ampla variedade de vertebrados.Studies on the behavioral and feeding habits of some species of phlebotominae sand flies have contributed to the comprehension of the epidemiology of leishmaniasis. In the present work, systematic captures were performed monthly in the municipality of Porteirinha (MG) using 28 light traps (CDC) from January to December 2002. Fourteen different species of phlebotomine were captured in a total of 1,408 specimens. The highest percentage of individuals (53.3%) was collected in the peridomicile against 46.7% in the intradomicile. Lutzomyia longipalpis was the predominant species in that region. The blood feeding of 38 females of this species from the field was analyzed by precipitin reaction. The results indicated that Lutzomyia longipalpis is an opportunist (65.1%) species that feeds on a wide variety of vertebrates in nature

Intradermal Immunization of <i>Leishmania donovani</i> Centrin Knock-Out Parasites in Combination with Salivary Protein LJM19 from Sand Fly Vector Induces a Durable Protective Immune Response in Hamsters

<div><p>Background</p><p>Visceral leishmaniasis (VL) is a neglected tropical disease and is fatal if untreated. There is no vaccine available against leishmaniasis. The majority of patients with cutaneous leishmaniasis (CL) or VL develop a long-term protective immunity after cure from infection, which indicates that development of an effective vaccine against leishmaniasis is possible. Such protection may also be achieved by immunization with live attenuated parasites that do not cause disease. We have previously reported a protective response in mice, hamsters and dogs with <i>Leishmania donovani</i> centrin gene knock-out parasites (<i>LdCen</i>^<i>-/-</i>), a live attenuated parasite with a cell division specific centrin1 gene deletion. In this study we have explored the effects of salivary protein LJM19 as an adjuvant and intradermal (ID) route of immunization on the efficacy of <i>LdCen</i>^<i>-/-</i> parasites as a vaccine against virulent <i>L</i>. <i>donovani</i>.</p><p>Methodology/Principal Findings</p><p>To explore the potential of a combination of <i>LdCen</i>^-/- parasites and salivary protein LJM19 as vaccine antigens, <i>LdCen</i>^-/- ID immunization followed by ID challenge with virulent <i>L</i>. <i>donovani</i> were performed in hamsters in a 9-month follow up study. We determined parasite burden (serial dilution), antibody production (ELISA) and cytokine expression (qPCR) in these animals. Compared to controls, animals immunized with <i>LdCen</i>^<i>-/-</i> + LJM19 induced a strong antibody response, a reduction in spleen and liver parasite burden and a higher expression of pro-inflammatory cytokines after immunization and one month post-challenge. Additionally, a low parasite load in lymph nodes, spleen and liver, and a non-inflamed spleen was observed in immunized animals 9 months after the challenge infection.</p><p>Conclusions</p><p>Our results demonstrate that an ID vaccination using <i>LdCen</i>^<i>-/-</i>parasites in combination with sand fly salivary protein LJM19 has the capability to confer long lasting protection against visceral leishmaniasis that is comparable to intravenous or intracardial immunization.</p></div

Parasite burden 9 months after challenge.

<p>Hamsters were challenged in the left ear with virulent <i>L</i>. <i>donovani</i> and presence of parasites was detected 9 months after challenge. The draining lymph node (A), spleen (B) and liver (C) were used in limiting dilution assay and data expressed as number of parasites/organ. Statistical differences (p<0.05) are indicated in letters (a: G1, b: G2 and c: G3). (D) Comparison of spleen size 9 months after challenge. The median of spleen size of hamsters of G1, G2, G3 and G4 was measured in centimeter. A representative sample from 6 immunized and challenged hamsters is shown.</p

Cytokine responses 5 weeks after immunization with <i>LdCen</i>^<i>-/-</i> parasites and LJM19.

<p>Immune response in the ear tissue of immunized hamsters was measured. The expression of mRNA encoding IFN-γ (A), iNOS (B), IL-12 (C), IL-4 (D) and IL-10 (E) was evaluated by qPCR. The data were normalized to β-Actin expression. (F) Fold increase of the cytokines compared to BSA (G4) group. Statistical differences (<i>p</i><0.05) are indicated in letters (a: G1).</p