RESUMO
Visceral leishmaniasis (VL) is a zoonosis caused by the protozoa of the genus Leishmania. Among the species, L. infantum and/or L. infantum (chagasi) are the most important species affecting the Americas. Domestic dogs are the main reservoir of the parasite and participate effectively in the parasite' transmission cycle. The Canine Visceral Leishmaniasis Control Program (PCLV) adopted in Brazil present as strategies the vector control, health education and serological diagnosis of CVL in dogs followed by culling of the seropositive ones. The resolution to eliminate seropositive dogs by euthanasia, when necessary, are the most controversial and least accepted by society. The diagnostic methods for canine visceral leishmaniasis, currently indicated and approved in Brazil by the Ministry of Health from Brazil are the Dual Path Platform (DPP)® as a screening test and the Enzyme immunoassay test (ELISA®). This study aimed to verify the presence of Leishmania spp. DNA in peripheral blood samples of dogs presenting positive serological results byDPP® and ELISA® tests,throughreal-time polymerase chain reaction (rt-PCR), using the pair of primers 150-152 already described. For this purpose, were collected blood samples from 185 seropositive dogs among them, 41 (22%) exhibited some clinical signal of disease, whereas 144 (78%) was asymptomatic. The animals were also analyzed according to gender, race and hair size. According to the results of rt-PCR, it was observed that among the185 seropositive dogs analyzed, only 132 (71%) presented positive results for CVL and 53 (29%) presented negative results. From this, 41/41 symptomatic dogs were positive (100%), while among the asymptomatic dogs, 91/144 were positive (63, 2%) and 53/144 were negative (36, 8%). Concerning the hair size of seropositive dogs, we found that 41 (22%) had long hair, while 144 (78%) had short hair. No statistical significance occurred between the results of rt-PCR, ELISA and DPP tests and the profile of the animals (gender, size of the dogs and hair size), probably due to the small number of samples and the sampling differences of each profile. But statistical significance occurred between the results of rt-PCR and the clinical evaluation, since the rt-PCR was positive in all symptomatic dogs. Thus, through these results, we reached at the following question, which may contribute to an important current debate: the dogs presenting CVL seropositive diagnosis confirmed by tests distributed by the Ministry of Health were in reality ill or were they seropositive by living in an endemic area of the disease? Would these asymptomatic seropositive dogs spread the disease to the inhabitants even presenting a low parasite charge circulating in the blood.
Assuntos
Doenças do Cão/diagnóstico , Leishmania/genética , Leishmania/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Anticorpos Antiprotozoários/sangue , Brasil , DNA de Protozoário/análise , Testes Diagnósticos de Rotina , Doenças do Cão/parasitologia , Cães , Feminino , Leishmania/patogenicidade , Leishmaniose Visceral/sangue , Masculino , Patologia Molecular , Testes Sorológicos/métodos , Testes Sorológicos/veterináriaRESUMO
BACKGROUND: The development of a vaccine for the prevention of visceral leishmaniasis (VL) still represents a significant unmet medical need. A human vaccine can be found if one takes into consideration that many people living in endemic areas of disease are infected but do not develop active VL, including those subjects with subclinical or asymptomatic infection. METHODS: In this study, a phage display was used to select phage-exposed peptides that were specific to immunoglobulin G (IgG) antibodies from asymptomatic and symptomatic VL patients, separating them from non-infected subjects. Phage clones presenting valid peptide sequences were selected and used as stimuli of peripheral blood mononuclear cells (PBMCs) obtained from both patients' groups and controls. Those with higher interferon-gamma (IFN-γ)/interleukin (IL)-10 ratios were further selected for vaccination tests. RESULTS: Among 17 evaluated clones, two were selected, B1 and D11, and used to immunize BALB/c mice in an attempt to further validate their in vivo protective efficacy against Leishmania infantum infection. Both clones induced partial protection against the parasite challenge, which was evidenced by the reduction of parasitism in the evaluated organs, a process mediated by a specific T helper (Th)1 immune response. CONCLUSIONS: To the best of our knowledge, this study is the first to use a rational strategy based on in vitro stimulation of human PBMCs with selected phage-displayed clones to obtain new immunogens against VL.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Leishmania infantum/imunologia , Leishmaniose Visceral/prevenção & controle , Vacinas Protozoárias/imunologia , Vacinas Protozoárias/isolamento & purificação , Células Th1/imunologia , Animais , Humanos , Imunoensaio , Interferon gama/metabolismo , Interleucina-10/metabolismo , Leishmaniose Visceral/imunologia , Programas de Rastreamento , Camundongos Endogâmicos BALB C , Biblioteca de PeptídeosRESUMO
In the present study, Leishmania braziliensis enolase was cloned and the recombinant protein (rEnolase) was evaluated for the serodiagnosis of canine and human visceral leishmaniosis (VL). For the canine VL diagnosis, this study examined serum samples of Leishmania infantum-infected dogs, from non-infected animals living in endemic or non-endemic areas of leishmaniosis, as well as those from Leish-Tec®-vaccinated dogs and Trypanosoma cruzi or Ehrlichia canis experimentally infected animals. For the human VL diagnosis, this study analyzed serum samples from VL patients, from non-infected subjects living in endemic or non-endemic areas of leishmaniosis, as well as those from T. cruzi-infected patients. In the results, an indirect ELISA method using rEnolase showed diagnostic sensitivity and specificity values of 100% and 98.57%, respectively, for canine VL serodiagnosis, and of 100% and 97.87%, respectively, for human VL diagnosis. These results showed rEnolase with an improved diagnostic performance when compared to the recombinant A2 protein, the crude soluble Leishmania antigenic preparation, and the recombinant K39-based immunochromatographic test. In conclusion, preliminary results suggest that the detection of antibodies against rEnolase improves the serodiagnosis of human and canine visceral leishmaniosis.
Assuntos
Doenças do Cão/sangue , Leishmania braziliensis/enzimologia , Leishmaniose Cutânea/veterinária , Fosfopiruvato Hidratase/sangue , Testes Sorológicos/veterinária , Adulto , Animais , Biomarcadores , Clonagem Molecular , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Cães , Feminino , Humanos , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/parasitologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes , Testes Sorológicos/métodos , Adulto JovemRESUMO
The serodiagnosis for tegumentary leishmaniasis (TL) presents problems related to the sensitivity and/or specificity of the tests. In the present study, an enzyme-linked immunosorbent assay (ELISA) technique was used to evaluate the performance from a Leishmania braziliensis hypothetical protein, LbHyM, in an attempt to compare its serological reactivity with a soluble Leishmania antigenic preparation (SLA) for the serodiagnosis of cutaneous (CL) and mucosal (ML) leishmaniasis. LbHyM was predicted to be a kinesin-like protein by bioinformatics tools. Serum samples were collected from both CL and ML patients, as well as from those with Chagas disease and from healthy subjects living in endemic or non-endemic areas of TL. Also, sera were collected from patients before and after the treatments, seeking to evaluate their serological follow-up in relation to the anti-protein and anti-parasite antibody levels. When an ELISA-rLbHyM assay was performed, it proved to be significantly more sensitive than ELISA-L. braziliensis SLA in detecting both CL and ML patients. Also, when using sera from Chagas disease patients, the ELISA-rLbHyM proved to be more specific than ELISA-SLA. The anti-protein and anti-parasite antibody levels were also evaluated 6 months after the treatments, and treated patients showed significantly lower levels of specific-rLbHyM antibodies, when compared to the anti-parasite antibody levels. In conclusion, the ELISA-rLbHyM assay can be considered a confirmatory serological technique for the serodiagnosis of L. braziliensis infection and can also be used in the serological follow-up of treated patients, aiming to correlate the low anti-protein antibody levels with the improvement of the healthy state of the patients.
Assuntos
Anticorpos Antiprotozoários/sangue , Doença de Chagas/diagnóstico , Cinesinas/imunologia , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/diagnóstico , Proteínas de Protozoários/imunologia , Adulto , Animais , Anticorpos Antiprotozoários/imunologia , Doença de Chagas/parasitologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Leishmania infantum/imunologia , Leishmaniose Cutânea/parasitologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Adulto JovemRESUMO
In the present study, two proteins cloned from Leishmania braziliensis species, a hypothetical protein (LbHyp) and the eukaryotic initiation factor 5a (EiF5a), were evaluated to protect BALB/c mice against L. amazonensis infection. The animals were immunized with the antigens, either separately or in combination, using saponin as an immune adjuvant in both cases. Spleen cells from vaccinated and later infected mice produced significantly higher levels of protein and parasite-specific IFN-γ, IL-12, and GM-CSF, in addition to low levels of IL-4 and IL-10. Evaluating the parasite load by means of a limiting dilution technique and quantitative Real-Time PCR, vaccinated animals presented significant reductions in the parasite load in both infected tissues and organs, as well as lower footpad swelling, when compared to the control (saline and saponin) groups. The best results regarding the protection of the animals were achieved when the combined vaccine was administered into the animals. Protection was associated with an IFN-γ production against parasite antigens, which was mediated by both CD4+ and CD8+ T cells and correlated with antileishmanial nitrite production. In conclusion, data from the present study show that this polyprotein vaccine, which combines two L. braziliensis proteins, can induce protection against L. amazonensis infection.
Assuntos
Antígenos de Protozoários/imunologia , Reações Cruzadas/imunologia , Leishmania braziliensis/imunologia , Leishmania mexicana/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Cutânea/prevenção & controle , Fatores de Iniciação de Peptídeos/imunologia , Proteínas de Ligação a RNA/imunologia , Animais , Antígenos de Protozoários/química , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Parasita/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos , Carga Parasitária , Fatores de Iniciação de Peptídeos/química , Proteínas de Ligação a RNA/química , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Vaccination can be considered the most cost-effective strategy to control neglected diseases, but nowadays there is not an effective vaccine available against leishmaniasis. In the present study, a vaccine based on the combination of the Leishmania-specific hypothetical protein (LiHyD) with saponin was tested in BALB/c mice against infection caused by Leishmania major and Leishmania braziliensis species. This antigen was firstly identified in Leishmania infantum and showed to be protective against infection of BALB/c mice using this parasite species. The immunogenicity of rLiHyD/saponin vaccine was evaluated, and the results showed that immunized mice produced high levels of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with rLiHyD, as well as by using L. major or L. braziliensis protein extracts. After challenge, vaccinated animals showed significant reductions in the infected footpad swellings, as well as in the parasite burden in the infection site, liver, spleen, and infected paws draining lymph nodes, when compared to those that were inoculated with the vaccine diluent (saline) or immunized with saponin. The immunization of rLiHyD without adjuvant was not protective against both challenges. The partial protection obtained by the rLiHyD/saponin vaccine was associated with a parasite-specific IL-12-dependent IFN-γ secretion, which was produced mainly by CD4(+) T cells. In these animals, a decrease in the parasite-mediated IL-4 and IL-10 responses, associated with the presence of high levels of LiHyD- and parasite-specific IgG2a isotype antibodies, were also observed. The present study showed that a hypothetical protein that was firstly identified in L. infantum, when combined to a Th1 adjuvant, was able to confer a cross-protection against highly infective stationary-phase promastigotes of two Leishmania species causing tegumentary leishmaniasis.
Assuntos
Leishmania braziliensis/imunologia , Leishmania infantum/imunologia , Leishmania major/imunologia , Vacinas contra Leishmaniose/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Citocinas/biossíntese , Feminino , Leishmaniose/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologia , VacinaçãoRESUMO
The present study aimed to evaluate a new Leishmania-specific hypothetical protein, LiHyT, as a vaccine candidate against VL. The immunogenicity of the recombinant protein (rLiHyT) plus saponin was evaluated in BALB/c mice. In the results, it is shown that rLiHyT plus saponin vaccinated mice produced high levels of IFN-γ, IL-12, and GM-CSF after in vitro stimulation of spleen cells using both rLiHyT and Leishmania infantum SLA. The protective efficacy was evaluated after subcutaneous challenge with stationary promastigotes of L. infantum. Immunized and infected mice, when compared to the controls, showed significant reductions in the number of parasites 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, mainly by CD4(+) T cells, with a minor contribution of CD8(+) T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 responses, as well as a predominance of LiHyT- and parasite-specific IgG2a isotype antibodies, were also observed. The present study showed that a new Leishmania-specific protein, when combined with a Th1-type adjuvant, presents potential to be used as a vaccine against VL.
Assuntos
Antígenos de Protozoários/imunologia , Leishmania infantum/imunologia , Leishmaniose Visceral/prevenção & controle , Vacinas Protozoárias/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Imunoglobulina G/sangue , Interleucina-4/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Saponinas/imunologiaRESUMO
In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4+ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite-specific IgG2a antibodies. The polyproteins vaccine administration induced a more pronounced Th1 response before and after challenge infection than individual vaccines, which was correlated to a higher control of parasite dissemination to internal organs.
Assuntos
Antígenos de Protozoários/uso terapêutico , Leishmania infantum/imunologia , Leishmaniose Visceral/prevenção & controle , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/uso terapêutico , Animais , Citocinas/metabolismo , Cães , Feminino , Imunidade Humoral , Leishmania infantum/crescimento & desenvolvimento , Camundongos Endogâmicos BALB C , Nitritos/metabolismo , Carga ParasitáriaRESUMO
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.
Assuntos
Biomarcadores , Técnicas de Visualização da Superfície Celular/métodos , Leishmaniose/diagnóstico , Leishmaniose/terapia , Vacinação , Animais , Biotecnologia , Descoberta de Drogas/métodos , Técnicas Genéticas , Humanos , Imunoterapia/métodos , Leishmaniose/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
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.
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