Phage-fused epitopes from Leishmania infantum used as immunogenic vaccines confer partial protection against Leishmania amazonensis infection

Two mimotopes of Leishmania infantum identified by phage display were evaluated as vaccine candidates in BALB/c mice against Leishmania amazonensis infection. The epitope-based immunogens, namely B10 and C01, presented as phagefused peptides; were used without association of a Th1 adjuvant, and they were administered isolated or in combination into animals. Both clones showed a specific production of interferon-gamma (IFN-γ), interleukin-12 (IL-12) and granulocyte/macrophage colony-stimulating factor (GM-CSF) after in vitro spleen cells stimulation, and they were able to induce a partial protection against infection. Significant reductions of parasite load in the infected footpads, liver, spleen, bone marrow and paws’ draining lymph nodes were observed in the immunized mice, in comparison with the control groups (saline, saponin, wild-type and non-relevant clones). Protection was associated with an IL-12-dependent production of IFN-γ, mediated mainly by CD8+ T cells, against parasite proteins. Protected mice also presented low levels of IL-4 and IL-10, as well as increased levels of parasite-specific IgG2a antibodies. The association of both clones resulted in an improved protection in relation to their individual use. More importantly, the absence of adjuvant did not diminish the cross-protective efficacy against Leishmania spp. infection. This study describes for the first time two epitope-based immunogens selected by phage display technology against L. infantum infected dogs sera, which induced a partial protection in BALB/c mice infected with L. amazonensisInstituto Nacional de Ciência e Tecnologia em Nanobiofarmacêutica (INCT-Nanobiofar), FAPEMIG (PRONEX APQ-0101909, CBB-APQ-00496-11 and CBB-APQ-00819-12), CAPES (Rede anobiotec/Brazil) and CNPq (APQ-472090/2011-9, APQ-482976/2012-8 and APQ-488237/2013-0). M.A.C.F.Peer Reviewe

Theranostic applications of phage display to control leishmaniasis: Selection of biomarkers for serodiagnostics, vaccination, and immunotherapy

© 2015, Sociedade Brasileira de Medicina Tropical. All rights reserved. Phage display is a high-throughput subtractive proteomic technology used for the generation and screening of large peptide and antibody libraries. It is based on the selection of phage-fused surface-exposed peptides that recognize specific ligands and demonstrate desired functionality for diagnostic and therapeutic purposes. Phage display has provided unmatched tools for controlling viral, bacterial, fungal, and parasitic infections, and allowed identification of new therapeutic targets to treat cancer, metabolic diseases, and other chronic conditions. This review presents recent advancements in serodiagnostics and prevention of leishmaniasis -an important tropical parasitic disease- achieved using phage display for the identification of novel antigens with improved sensitivity and specificity. Our focus is on theranostics of visceral leishmaniasis with the aim to develop biomarker candidates exhibiting both diagnostic and therapeutic potential to fight this important, yet neglected, tropical disease.Peer Reviewe

An optimized nanoparticle delivery system based on chitosan and chondroitin sulfate molecules reduces the toxicity of amphotericin B and is effective in treating tegumentary leishmaniasis

Amphotericin B (AmpB) is active against leishmaniasis, but its use is hampered due to its high toxicity observed in patients. In this study, a nanoparticles-delivery system for AmpB (NQC-AmpB), containing chitosan and chondroitin sulfate molecules, was evaluated in BALB/c mice against Leishmania amazonensis. An in vivo biodistribution study, including biochemical and toxicological evaluations, was performed to evaluate the toxicity of AmpB. Nanoparticles were radiolabeled with technetium-99m and injected in mice. The products presented a similar biodistribution in the liver, spleen, and kidneys of the animals. Free AmpB induced alterations in the body weight of the mice, which, in the biochemical analysis, indicated hepatic and renal injury, as well as morphological damage to the kidneys of the animals. In general, no significant organic alteration was observed in the animals treated with NQC-AmpB. Mice were infected with L. amazonensis and treated with the nanoparticles or free AmpB; then, parasitological and immunological analyses were performed. The NQC-AmpB group, as compared to the control groups, presented significant reductions in the lesion size and in the parasite burden in all evaluated organs. These animals presented significantly higher levels of IFN-γ and IL-12, and low levels of IL-4 and IL-10, when compared to the control groups. The NQC-AmpB system was effective in reducing the infection in the animals, and proved to be effective in diminishing the toxicity evoked by AmpB, which was observed when it was administered alone. In conclusion, NQC-AmpB could be considered a viable possibility for future studies in the treatment of leishmaniasisThis work was supported by grants from Pró-Reitoria de Pesquisa from UFMG (Edital 01/2014), Instituto Nacional de Ciência e Tecnologia em Nano-biofarmacêutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-00496-11 and CBB-APQ-00819-12), and CNPq (APQ-472090/2011-9 and APQ-482976/2012-8). MACF is a grant recipient of FAPEMIG/CAPES. EAFC, VNC, and AAGF are grant recipients of CNPq. Eduardo AF Coelho and André AG Faraco are co-senior authors of this stud

INCIDÊNCIA DE CONSUMO DE BEBIDA ALCOÓLICA ENTRE AS VÍTIMAS DE SUICÍDIO NO DISTRITO FEDERAL

Suicide is motivated by multiple causes, and the abusive consumption of alcoholic beverages can be an important sign that individuals do not have physical and emotional control of themselves. To estimate the incidence of alcohol consumption, immediately before suicide, among people who died in 2018 in the Brazilian Federal District. This is a population-based study, of a descriptive and exploratory nature, of all deaths which occurred in the Brazilian Federal District from January 1 to December 31, 2018. Data were collected from the Mortality Information System and Police reports. The incidence and respective 95% confidence intervals were calculated. This study was approved by the Research Ethics Committee. The incidence of alcohol consumption just before the suicide was 18.6% (CI 95%: 18.56% to 18.62%). Males, blacks, trade workers, those without a partner, young people, and individuals with less than 8 years of study had the highest indicators. There was a greater percentage of alcohol consumption in the fall/winter, at the end of the month, in rural areas, in people who had never attempted suicide before and when the modus operandi was choking or hanging. The consumption of drugs, such as methamphetamine, ecstasy, amphetamine, and cannabis, was observed together with alcoholic beverages. The results of this research signaled an important public health problem, which is the use of alcohol to potentially encourage suicide. Effective public policies to tackle the abusive consumption of alcoholic beverages and suicide are necessary.O suicídio é motivado por múltiplas causas e o consumo abusivo de bebidas alcoólicas pode ser um sinal importante de que os indivíduos não têm controle físico e emocional de si mesmos. Estimar a incidência do consumo de álcool, imediatamente antes do suicídio, entre as pessoas que morreram em 2018 no Distrito Federal. Trata-se de um estudo de base populacional, de caráter descritivo e exploratório, de todos os óbitos ocorridos no Distrito Federal de 1º de janeiro a 31 de dezembro de 2018. Os dados foram coletados do Sistema de Informações sobre Mortalidade e relatórios policiais. Foram calculados os intervalos de confiança de 95%. Este estudo foi aprovado pelo Comitê de Ética em Pesquisa. A incidência de consumo de álcool pouco antes do suicídio foi de 18,6% (IC 95%: 18,56% a 18,62%). Homens, negros, trabalhadores do comércio, aqueles sem parceiro, jovens e indivíduos com menos de 8 anos de estudo apresentaram os maiores indicadores. Houve maior percentual de consumo de álcool no outono/inverno, no final do mês, em áreas rurais, em pessoas que nunca haviam tentado suicídio antes e quando o modus operandi estava engasgando ou pendurado. O consumo de drogas, como metanfetamina, ecstasy, anfetamina e cannabis, foi observado juntamente com bebidas alcoólicas. Os resultados desta pesquisa sinalizaram um importante problema de saúde pública, que é o uso de álcool para potencialmente incentivar o suicídio. São necessárias políticas públicas eficazes para enfrentar o consumo abusivo de bebidas alcoólicas e o suicídio.El suicidio está motivado por múltiples causas, y el consumo abusivo de bebidas alcohólicas puede ser una señal importante de que las personas no tienen control físico y emocional de sí mismos. Estimar la incidencia del consumo de alcohol, inmediatamente antes del suicidio, entre las personas que murieron en 2018 en el Distrito Federal de Brasil. Se trata de un estudio poblacional, de carácter descriptivo y exploratorio, de todas las muertes ocurridas en el Distrito Federal de Brasil del 1 de enero al 31 de diciembre de 2018. Los datos se recopilaron del Sistema de Información de Mortalidad y de los informes de la Policía. Se calcularon la incidencia y los respectivos intervalos de confianza del 95%. Este estudio fue aprobado por el Comité de ética de la investigación. La incidencia del consumo de alcohol justo antes del suicidio fue del 18,6% (CI 95%: 18,56% a 18,62%). Los hombres, los negros, los trabajadores del comercio, los que no tienen pareja, los jóvenes y los individuos con menos de 8 años de estudio tenían los indicadores más altos. Había un mayor porcentaje de consumo de alcohol en otoño/invierno, a finales de mes, en las zonas rurales, en personas que nunca habían intentado suicidarse antes y cuando el modus operandi se estaba asfixiando o ahorcando. El consumo de drogas, como la metanfetamina, el éxtasis, la anfetamina y el cannabis, se observó junto con las bebidas alcohólicas. Los resultados de esta investigación indicaron un importante problema de salud pública, que es el uso del alcohol para fomentar potencialmente el suicidio. Las políticas públicas eficaces para hacer frente al consumo abusivo de bebidas alcohólicas y el suicidio so

INCIDÊNCIA DE CONSUMO DE BEBIDA ALCOÓLICA ENTRE AS VÍTIMAS DE SUICÍDIO NO DISTRITO FEDERAL

Suicide is motivated by multiple causes, and the abusive consumption of alcoholic beverages can be an important sign that individuals do not have physical and emotional control of themselves. To estimate the incidence of alcohol consumption, immediately before suicide, among people who died in 2018 in the Brazilian Federal District. This is a population-based study, of a descriptive and exploratory nature, of all deaths which occurred in the Brazilian Federal District from January 1 to December 31, 2018. Data were collected from the Mortality Information System and Police reports. The incidence and respective 95% confidence intervals were calculated. This study was approved by the Research Ethics Committee. The incidence of alcohol consumption just before the suicide was 18.6% (CI 95%: 18.56% to 18.62%). Males, blacks, trade workers, those without a partner, young people, and individuals with less than 8 years of study had the highest indicators. There was a greater percentage of alcohol consumption in the fall/winter, at the end of the month, in rural areas, in people who had never attempted suicide before and when the modus operandi was choking or hanging. The consumption of drugs, such as methamphetamine, ecstasy, amphetamine, and cannabis, was observed together with alcoholic beverages. The results of this research signaled an important public health problem, which is the use of alcohol to potentially encourage suicide. Effective public policies to tackle the abusive consumption of alcoholic beverages and suicide are necessary.O suicídio é motivado por múltiplas causas e o consumo abusivo de bebidas alcoólicas pode ser um sinal importante de que os indivíduos não têm controle físico e emocional de si mesmos. Estimar a incidência do consumo de álcool, imediatamente antes do suicídio, entre as pessoas que morreram em 2018 no Distrito Federal. Trata-se de um estudo de base populacional, de caráter descritivo e exploratório, de todos os óbitos ocorridos no Distrito Federal de 1º de janeiro a 31 de dezembro de 2018. Os dados foram coletados do Sistema de Informações sobre Mortalidade e relatórios policiais. Foram calculados os intervalos de confiança de 95%. Este estudo foi aprovado pelo Comitê de Ética em Pesquisa. A incidência de consumo de álcool pouco antes do suicídio foi de 18,6% (IC 95%: 18,56% a 18,62%). Homens, negros, trabalhadores do comércio, aqueles sem parceiro, jovens e indivíduos com menos de 8 anos de estudo apresentaram os maiores indicadores. Houve maior percentual de consumo de álcool no outono/inverno, no final do mês, em áreas rurais, em pessoas que nunca haviam tentado suicídio antes e quando o modus operandi estava engasgando ou pendurado. O consumo de drogas, como metanfetamina, ecstasy, anfetamina e cannabis, foi observado juntamente com bebidas alcoólicas. Os resultados desta pesquisa sinalizaram um importante problema de saúde pública, que é o uso de álcool para potencialmente incentivar o suicídio. São necessárias políticas públicas eficazes para enfrentar o consumo abusivo de bebidas alcoólicas e o suicídio.El suicidio está motivado por múltiples causas, y el consumo abusivo de bebidas alcohólicas puede ser una señal importante de que las personas no tienen control físico y emocional de sí mismos. Estimar la incidencia del consumo de alcohol, inmediatamente antes del suicidio, entre las personas que murieron en 2018 en el Distrito Federal de Brasil. Se trata de un estudio poblacional, de carácter descriptivo y exploratorio, de todas las muertes ocurridas en el Distrito Federal de Brasil del 1 de enero al 31 de diciembre de 2018. Los datos se recopilaron del Sistema de Información de Mortalidad y de los informes de la Policía. Se calcularon la incidencia y los respectivos intervalos de confianza del 95%. Este estudio fue aprobado por el Comité de ética de la investigación. La incidencia del consumo de alcohol justo antes del suicidio fue del 18,6% (CI 95%: 18,56% a 18,62%). Los hombres, los negros, los trabajadores del comercio, los que no tienen pareja, los jóvenes y los individuos con menos de 8 años de estudio tenían los indicadores más altos. Había un mayor porcentaje de consumo de alcohol en otoño/invierno, a finales de mes, en las zonas rurales, en personas que nunca habían intentado suicidarse antes y cuando el modus operandi se estaba asfixiando o ahorcando. El consumo de drogas, como la metanfetamina, el éxtasis, la anfetamina y el cannabis, se observó junto con las bebidas alcohólicas. Los resultados de esta investigación indicaron un importante problema de salud pública, que es el uso del alcohol para fomentar potencialmente el suicidio. Las políticas públicas eficaces para hacer frente al consumo abusivo de bebidas alcohólicas y el suicidio so

Mimotope-based vaccines of leishmania infantum antigens and their protective efficacy against visceral leishmaniasis

© 2014 Costa et al. The development of cost-effective prophylactic strategies to prevent leishmaniasis has become a high-priority. The present study has used the phage display technology to identify new immunogens, which were evaluated as vaccines in the murine model of visceral leishmaniasis (VL). Epitope-based immunogens, represented by phage-fused peptides thatimic Leishmania infantum antigens, were selected according to their affinity to antibodies from asymptomatic and symptomatic VL dogs' sera. Methodology/Main Findings: Twenty phage clones were selected after three selection cycles, and were evaluated by means of in vitro assays of the immune stimulation of spleen cells derived from naive and chronically infected with L. infantum BALB/c mice. Clones that were able to induce specific Th1 immune response, represented by high levels of IFN-c and low levels of IL-4 were selected, and based on their selectivity and specificity, two clones, namely B10 and C01, were further employed in the vaccination protocols. BALB/c mice vaccinated with clones plus saponin showed both a high and specific production of IFN-c, IL-12, and GM-CSF after in vitro stimulation with individual clones or L. infantum extracts. Additionally, these animals, when compared to control groups (saline, saponin, wild-type phage plus saponin, or nonrelevant phage clone plus saponin), showed significant reductions in the parasite burden in the liver, spleen, bone marrow, and paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-c, mainly by CD8+ T cells, against parasite proteins. These animals also presented decreased parasite-mediated IL-4 and IL-10 responses, and increased levels of parasite-specific IgG2a antibodies. Conclusions/Significance: This study describes two phage clones that mimic L. infantum antigens, which were directly used as immunogens in vaccines and presented Th1-type immune responses, and that significantly reduced the parasite burden. This is the first study that describes phage-displayed peptides as successful immunogens in vaccine formulations against VL.Instituto Nacional de Cieˆncia e Tecnologia em Nanobiofarmaceutica (INCT-Nanobiofar), FAPEMIG (PRONEX APQ-0101909, CBB-APQ-00496-11 and CBB-APQ-00819-12), CAPES (Rede Nanobiotec/Brasil) and CNPq (APQ-472090/2011-9 and APQ- 482976/2012-8)Peer Reviewe

Subtractive phage display selection from canine visceral leishmaniasis identifies novel epitopes that mimic leishmania infantum antigens with potential serodiagnosis applications

Visceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy and Trypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected with Leishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n = 31) compared to those from vaccinated dogs (n = 21), experimentally infected dogs with cross-reactive parasites (n = 23), and healthy controls (n = 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity with T. cruzi- or Ehrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes of L. infantum antigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs. Copyright © 2014, American Society for Microbiology. All Rights Reserved.Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica (INCT Nano-Biofar), Rede Nanobiotec/Brasil-UFU (CAPES), PRONEX-FAPEMIG (APQ-01019-09), FAPEMIG (APQ-00496-11 and APQ-00819-12), and CNPq (APQ-472090/2011-9 and APQ-482976/2012-8)Peer Reviewe

Antigenicity and Protective Efficacy of a Leishmania Amastigote-specific Protein, Member of the Super-oxygenase Family, against Visceral Leishmaniasis

Background:The present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1), previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL.Methodology/Principal Findings:The LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1) was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin), showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed.Conclusions/Significance:The present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL. © 2013 Martins et al.UFMG (Edital 07/2012), Instituto Nacional de Ciencia e Tecnologia em Nanobiofarmace utica; Fundacao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG; CBB-APQ-02364-08, PPSUS/MS/CNPq/FAPEMIG/SES-MG/CBB-APQ-00356-10, and CBB-APQ-00496-11); Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq; APQ-472090/2011-9); Instituto Nacional de Ciencia e Tecnologia; FAPEMIG/CAPES; Ministerio de Ciencia e Innovacion (FIS/PI1100095)Peer Reviewe

Evaluation of parasitological and immunological parameters of Leishmania chagasi infection in BALB/c mice using different doses and routes of inoculation of parasites

Experimental vaccines to protect against visceral leishmaniasis (VL) have been developed by using BALB/c mice infected with a large (107 to 108) inoculum of parasites. Remarkably, prior literature has reported that the poor protection observed is mainly due to the high susceptibility of this strain. To determine factors inherent to mice that might abrogate vaccine-induced efficacy, the present research sought to investigate the impact of the administration of different infective inoculums of Leishmania chagasi (syn. L. infantum) in BALB/c mice, evaluating subcutaneous and intravenous routes of administration as well as parasitological and immunological parameters over different periods of time. This study shows that the injection of a highly infective inoculum in mice, through both subcutaneous and intravenous routes, results in a sustained infection. The mice developed a high parasite load in the liver; however, these values diminished over time. This result did not corroborate with the parasite load in the bone marrow and brain and proved to be expressively different in the spleen and draining lymph nodes, where the values increased over time. Mice infected with a low dose of parasites (103) showed a certain resistance against infection, based mainly on the IFN-γ and oxide nitric production. Considering all the elements, it could be concluded that the models employing high doses (107) of L. chagasi in BALB/c mice can bring about an imbalance in the animals’ immune response, thus allowing for the development of the disease at the expense of efficacy within the vaccine candidates.This study was supported by grants from Pró- Reitoria de Pesquisa from UFMG (Edital 08/2011), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG; CBB-APQ-01322- 08), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; APQ-577483/2008-0), and Instituto Nacional de Ciência e Tecnologia em Nanobiofarmacêutica (INCT/Nano-BIOFAR), CNPq. DGV and EAFC are grant recipients of CNPq, while DMO, MAFC, and MACF are grant recipients of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).Peer reviewe