Expression of anti-Z-DNA single chain antibody variable fragment on the filamentous phage surface

We describe the expression of an anti-Z-DNA single chain variable region antibody fragment (scFv) on a filamentous phage surface. Four vectors for phage display were constructed. Two of them are able to display multiple copies of the antibody fragment, and the others can be used to make monovalent libraries. The vectors use different promoter/leader sequences to direct the expression of the fused proteins. All were able to promote the assembly of fusion virion particles. In this paper we also show the affinity selection (biopanning) of those phage-antibodies based on the capacity of their products to recognize the antigen. We used biotinylated Z-DNA and the selection was performed in a solution phase fashion. The data presented here indicate that these vectors can be further used to construct anti-nucleic acid antibody fragment libraries that can be used to study the basis of nucleic acid-protein interaction and its role in autoimmunity mechanisms

Optimization of heterologous protein production in Chinese hamster ovary cells under overexpression of spliced form of human X-box binding protein

Background: The optimization of protein production is a complex and challenging problem in biotechnology. Different techniques for transcription, translation engineering and the optimization of cell culture conditions have been used to improve protein secretion, but there remain many open problems involving post-translational modifications of the secreted protein and cell line stability. Results: In this work, we focus on the regulation of secreted protein specific productivity (using a recombinant human immunoglobulin G (IgG)) by controlling the expression of the spliced form of human X-box binding protein (XBP-(s)) in Chinese hamster ovary cells (CHO-K1) under doxycycline (DOX) induction at different temperatures. We observed a four-fold increase in specific IgG productivity by CHO cells under elevated concentrations of DOX at 30°C compared to 37°C, without detectable differences in binding activity in vitro or changes in the structural integrity of IgG. In addition, we found a correlation between the overexpression of human XBP-1(s) (and, as a consequence, endoplasmic reticulum (ER) size expansion) and the specific IgG productivity under DOX induction. Conclusions: Our data suggest the T-REx system overexpressing human XBP-1(s) can be successfully used in CHO-K1 cells for human immunoglobulin production

The efficacy of humanized antibody against the Sporothrix antigen, gp70, in promoting phagocytosis and reducing disease burden

Sporotrichosis is a subcutaneous mycosis distributed worldwide and is frequently reported in countries with tropical climates, as Latin America countries. We previously demonstrated that mice with sporotrichosis produce specific antibodies against a 70-kDa fungal protein, indicating that specific antibodies against this molecule may help to control the sporotrichosis. IgG1 monoclonal antibody was generated, and called mAbP6E7, in mice against a 70-kDa glycoprotein (gp70) of S. schenckii. The mAbP6E7 showed prophylactic and therapeutic activity against sporotrichosis. However, this antibody has a murine origin, and this can generate an immune response when administered to humans, precluding its use for a prolonged time. For its possible use in the treatment of human sporotrichosis, we humanized the mAbP6E7 by genetic engineering. Once expressed, the humanized antibodies had good stability and were able to bind to the 70-kDa cell wall antigens of Sporothrix schenckii and S. brasiliensis. The humanized P6E7 were able to opsonize S. schenckii yeasts, thus increasing the phagocytic index in human monocyte-derived macrophages. The treatment with humanized P6E7 decreased fungal burden in vivo. These data suggest that humanized P6E7 may have a therapeutic role in sporotrichosis

Development and characterization of recombinant antibody Fragments that recognize and neutralize in vitro Stx2 toxin from shiga toxin-producing escherichia coli

Background: Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. Methods: and Findings In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. Conclusion: In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro

HSF-1, HIF-1and HSP90 expression on recombinant Pichia pastoris under fed-batch fermentation

Pichia pastoris is a methylotrophic yeast used as an efficient expression system for heterologous protein production as compared to other expression systems. Considering that every cell must respond to environmental changes to survive and differentiate, determination of endogenous protein related to heat stress responses and hypoxia, it would necessary to establish the temperature and methanol concentration conditions for optimal growth. The aim of this study is characterize the culture conditions through the putative biomarkers in different conditions of temperature and methanol concentration. Three yeast cultures were performed: 3X = 3% methanol -10 °C, 4X = 3% methanol -30 °C, and 5X = 1% methanol -10 °C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. The western blot results of HIF-1α and HSP-90 did not indicate statistically significant in the culture conditions studied. Respect to biomarkers location, HIF-1α and HSP-90 presented differences between cultures. In conclusion, the results suggest the cultures in a hypoxic condition produce a high density and yeast cells smaller. Beside the high density would not necessary related with a high production of recombinant proteins in modified-genetically P. pastoris

Phage Display as a Strategy to Obtain Anti-flavivirus Monoclonal Antibodies

Arbovirus of the Flaviviridae family represents an issue worldwide, particularly because it can lead to serious illness and death in some countries. There is still a great complexity in obtaining effective therapies and specific and sensitive diagnostic tests, due to the high antigenic similarity between them. This similarity may account for antibodies cross reactivity which has positive and negative consequences for the course of infectious diseases. Among dengue virus (DENV) serotype infections, the cross-reactivity can increase virus replication and the risk of a severe disease by a mechanism known as an antibody-dependent enhancement (ADE). The search for serological biomarkers through monoclonal antibodies (MAbs) that identify unique viral regions can assist in the differential detection, whereas the development of recombinant antibodies with a neutralizing potential can lead to the establishment of efficacious treatments. The Phage Display methodology emerged as one of the main alternatives for the selection of human MAbs with high affinity for a specific target. Therefore, this technology can be a faster alternative for the development of specific diagnostic platforms and efficient and safe treatments for flavivirus infections. In this context, we propose for this chapter a discussion about Phage Display as a strategy to obtain MAbs for DENV and other flaviviruses

Molecular Detection of Medically Important Candida species from Droppings of Pigeons (Columbiformes) and Captive Birds (Passeriformes and Psittaciformes)

Passeriformes and Psittaciformes birds and pigeons (Columba livia) are known to be reservoirs of microorganisms, and their stool allows fungi development. Since accumulated avian excreta can interfere with public health, this study aimed to perform a molecular screening of medically important Candida species in pigeon droppings in public places and birds raised in captivity. Excreta collected from captive birds (3 residences) and pigeons (4 districts) were inoculated on Sabouraud dextrose agar with chloramphenicol for Gram staining and subculture on Hicrome® Candida. Three DNA extraction methods were performed for comparison (commercial kit, in-house and by boiling) and PCR to screen 6 clinically important Candida species among the isolates. The correlation between phenotypic and molecular methods was calculated by kappa/K. Only 6 C. parapsilosis (20%) were identified from captive birds’ feces among 30 isolates (80% not identified), while pigeons’ feces harbored a greater diversity, with the 6 pathogenic species confirmed among 41 isolates: C. albicans (31.70%/13), C. krusei (14.63%/6), C. tropicalis (14.63%/6), C. parapsilosis (17.10%/7), C. glabrata (14.63%/6) and C. guilliermondii (7.31%/3); 100% correlation between tested methods (K = 1) for the first 3 species. Boiling DNA extraction method was fast and efficient to obtain viable DNA from Candida spp. for PCR. Our results indicate that pigeon droppings harbor more potentially pathogenic species than birds in residential captivity, which probably have non-albicans Candida less frequently isolated in infectious processes. The greater availability of nutrients may have contributed to a diversity of Candida spp. in feces from public environments.</p

A integração do Arca - Repositório Institucional da Fiocruz com a Plataforma de Ciência de Dados aplicada à Saúde

Apresenta o projeto desenvolvido entre o Laboratório de Ciência de Dados aplicada À Saúde, do Instituto de Informação Científica e Tecnológica em Saúde (ICICT) e o Arca – Repositório Institucional da Fiocruz. O projeto teve como objetivos: melhorar a curadoria dos dados inseridos no repositório institucional, visando a qualidade das informações, e a recuperação e a visualização de dados, oferecendo uma plataforma que permite a extração de informações com potencial de uso pela gestão e pela pesquisa. No processo de curadoria foi possível identificar inconsistências no preenchimento dos metadados, utilizando classificação automática e machine learning, e consequente correção, de forma a garantir a qualidade das informações e dos dados extraídos. Outro fator importante para a realização do projeto foi a utilização do software Kibana e do Elasticsearch para a visualização de dados de forma dinâmica, oferecendo uma plataforma de exploração interativa para extração e mineração de dados. O software permitiu a utilização de filtros e combinações de dados contidos no Arca, como produção por tipo de material, Unidades da Fiocruz, assunto, autor, ano e direito autoral de forma que possam ser manipulados pelas diferentes unidades/comunidades representadas no Repositório Institucional.Fundação Oswaldo Cru

A integração do Arca - Repositório Institucional da Fiocruz com a Plataforma de Ciência de Dados aplicada à Saúde

Apresenta o projeto desenvolvido entre o Laboratório de Ciência de Dados aplicada À Saúde, do Instituto de Informação Científica e Tecnológica em Saúde (ICICT) e o Arca – Repositório Institucional da Fiocruz. O projeto teve como objetivos: melhorar a curadoria dos dados inseridos no repositório institucional, visando a qualidade das informações, e a recuperação e a visualização de dados, oferecendo uma plataforma que permite a extração de informações com potencial de uso pela gestão e pela pesquisa. No processo de curadoria foi possível identificar inconsistências no preenchimento dos metadados, utilizando classificação automática e machine learning, e consequente correção, de forma a garantir a qualidade das informações e dos dados extraídos. Outro fator importante para a realização do projeto foi a utilização do software Kibana e do Elasticsearch para a visualização de dados de forma dinâmica, oferecendo uma plataforma de exploração interativa para extração e mineração de dados. O software permitiu a utilização de filtros e combinações de dados contidos no Arca, como produção por tipo de material, Unidades da Fiocruz, assunto, autor, ano e direito autoral de forma que possam ser manipulados pelas diferentes unidades/comunidades representadas no Repositório Institucional.Fundação Oswaldo Cru

A integração do Arca - Repositório Institucional da Fiocruz com a Plataforma de Ciência de Dados aplicada à Saúde

Apresenta o projeto desenvolvido entre o Laboratório de Ciência de Dados aplicada À Saúde, do Instituto de Informação Científica e Tecnológica em Saúde (ICICT) e o Arca – Repositório Institucional da Fiocruz. O projeto teve como objetivos: melhorar a curadoria dos dados inseridos no repositório institucional, visando a qualidade das informações, e a recuperação e a visualização de dados, oferecendo uma plataforma que permite a extração de informações com potencial de uso pela gestão e pela pesquisa. No processo de curadoria foi possível identificar inconsistências no preenchimento dos metadados, utilizando classificação automática e machine learning, e consequente correção, de forma a garantir a qualidade das informações e dos dados extraídos. Outro fator importante para a realização do projeto foi a utilização do software Kibana e do Elasticsearch para a visualização de dados de forma dinâmica, oferecendo uma plataforma de exploração interativa para extração e mineração de dados. O software permitiu a utilização de filtros e combinações de dados contidos no Arca, como produção por tipo de material, Unidades da Fiocruz, assunto, autor, ano e direito autoral de forma que possam ser manipulados pelas diferentes unidades/comunidades representadas no Repositório Institucional.Fundação Oswaldo Cru