Seleção de antígenos de Leishmania infantum por imunoproteômica para emprego no diagnóstico da leishmaniose visceral humana / Selection of Leishmania infantum antigens by immunoproteomics for use in the diagnosis of human visceral leishmaniasis

O controle da leishmaniose visceral (LV) requer um diagnóstico e tratamento adequados, uma vez que o diagnóstico preciso é fundamental para um regime medicamentoso eficaz para os pacientes. Nesse contexto, as ferramentas biotecnológicas devem ser aprimoradas para o manejo clínico e a avaliação epidemiológica da doença. No entanto, existem limitações relacionadas com a sensibilidade e/ou especificidade dos antígenos usados atualmente, mostrando a necessidade de identificação de novas moléculas para serem testadas em um diagnóstico sorológico mais sensível e específico. Neste sentido, no presente estudo, uma abordagem imunoproteômica foi usada para identificar proteínas antigênicas das formas promastigotas e amastigotas da espécie Leishmania infantum, causadora de LV em nosso país, por meio de seu reconhecimento por anticorpos em soros de pacientes com a doença. Amostras de indivíduos saudáveis residentes em região endêmica da doença e de pacientes com Doença de Chagas foram utilizadas com a função de se obter proteínas mais específicas ao parasito Leishmania para serem avaliadas no diagnóstico da LV. Como resultados obtidos, um total de 29 e 21 proteínas foram identificadas nos extratos de formas promastigotas e amastigotas dos parasitos, respectivamente. Para a validação da capacidade diagnóstica, duas proteínas, endonuclease III e GTP-binding protein, foram selecionadas, clonadas, expressas e purificadas para serem testadas em experimentos de ELISA. Os resultados dos testes mostraram valores de sensibilidade e especificidade superiores a 99,0% para a identificação da LV. Os antígenos ainda exibiram um diferencial ao apresentarem baixa reatividade sorológica em pacientes curados e tratados, sugerindo a possibilidade de que as mesmas possam ser aplicadas como marcadores prognósticos da doença. Em conclusão, o estudo imunoproteômico se mostrou eficaz na seleção de proteínas antigênicas de L. infantum e duas delas, endonuclease III e GTP-binding protein, foram bem avaliadas para o diagnóstico da LV frente a um painel sorológico, além de demonstrarem um potencial para monitoramento de pacientes com LV após o tratamento.

The control of visceral leishmaniasis (VL) requires an adequate diagnosis and treatment, since an accurate diagnosis is essential for an effective medication regimen for patients. In this context, biotechnological tools must be improved for the clinical management and epidemiological assessment of the disease. However, there are limitations related to the sensitivity and / or specificity of the antigens currently used, showing the necessity to identify new molecules to be tested in a more sensitive and specific serological diagnosis. In this sense, in the present study, an immunoproteomics approach was used to identify antigenic proteins of the Leishmania infantum promastigote and amastigote forms, which causes VL in our country, through its recognition by antibodies in sera of patients with the disease. Samples from healthy individuals living in an endemic region of the disease and from patients with Chagas disease were used to obtain more specific proteins for the Leishmania parasite, aiming their future application in the VL diagnosis. As results obtained, a total of 29 and 21 proteins were identified in the extracts of parasitic promastigotes and amastigotes, respectively. For validation of the diagnostic capacity, two proteins, endonuclease III and GTP-binding protein, were selected, cloned, expressed and purified to be tested in ELISA experiments. The test results showed sensitivity and specificity values greater than 99.0% for the identification of VL. The antigens also exhibited a differential when presenting low serological reactivity in cured and treated patients, suggesting the possibility that they can be applied as prognostic markers of the disease. In conclusion, the immunoproteomic study proved to be effective in the selection of L. infantum antigenic proteins and two of them, endonuclease III and GTP-binding protein, were well evaluated for the diagnosis of VL against a serological panel, in addition, demonstrating a potential for monitoring patients with VL after treatment.

Potential Vaccine Targets against Rabbit Coccidiosis by Immunoproteomic Analysis

The aim of this study was to identify antigens for a vaccine or drug target to control rabbit coccidiosis. A combination of 2-dimensional electrophoresis, immunoblotting, and mass spectrometric analysis were used to identify novel antigens from the sporozoites of Eimeria stiedae. Protein spots were recognized by the sera of New Zealand rabbits infected artificially with E. stiedae. The proteins were characterized by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) analysis in combination with bioinformatics. Approximately 868 protein spots were detected by silver-staining, and a total of 41 immunoreactive protein spots were recognized by anti-E. stiedae sera. Finally, 23 protein spots were successfully identified. The proteins such as heat shock protein 70 and aspartyl protease may have potential as immunodiagnostic or vaccine antigens. The immunoreactive proteins were found to possess a wide range of biological functions. This study is the first to report the proteins recognized by sera of infected rabbits with E. stiedae, which might be helpful in identifying potential targets for vaccine development to control rabbit coccidiosis.

Immunoproteomic analysis of outer membrane proteins of Aeromonas hydrophila strain ATCC7966 / 中华微生物学和免疫学杂志

Objective To identify highly sensitive and specific antigens in Aeromonas hydrophila strain ATCC7966 by using immunoproteomics.Methods Outer membrane proteins were extracted from the Aeromonas hydrophila strain by using two-dimension electrophoresis and identified by LC-LTQ-XL-MS ( liq-uid chromatography coupled with linear ion trap mass spectrometry).Western blot assay was performed to screen out the immunogenic proteins.Results A total of 43 peptides representing 39 proteins were identi-fied by LC-LTQ-XL-MS.Among the 39 proteins, 69% were outer membrane proteins and 12% were inner membrane proteins.They were involved in the process of transportation, cell motility, biosynthesis, etc. One candidate vaccine antigen was identified by using two-dimensional Western blot analysis .Conclusion The immunoproteomics approach could be used to identify immunogenic proteins of Aeromonas hydrophila. This study provided references for further investigation on candidate vaccine antigens.

Progress of research on the proteomics of Bacillus anthracis / 军事医学科学院院刊

Proteomics, which has been widely used in life science, is an emerging discipline following genomics. It can help to explore the pathogenic mechanism and early onset marker of Bacillus anthracis, playing an important part in the prevention, diagnosis and treatment of B.anthracis. In this paper,the application of proteomics in the research of B.anthracis is reviewed.