Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-05-10T16:21:40Z No. of bitstreams: 1 Jessica Mariane Ferreira Mendes Produção... 2016.pdf: 1965926 bytes, checksum: ac5119592b97390470f4914f6fcbddf0 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-05-10T16:21:54Z (GMT) No. of bitstreams: 1 Jessica Mariane Ferreira Mendes Produção... 2016.pdf: 1965926 bytes, checksum: ac5119592b97390470f4914f6fcbddf0 (MD5) / Made available in DSpace on 2016-05-10T16:21:54Z (GMT). No. of bitstreams: 1 Jessica Mariane Ferreira Mendes Produção... 2016.pdf: 1965926 bytes, checksum: ac5119592b97390470f4914f6fcbddf0 (MD5) Previous issue date: 2016 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Uma vacina efetiva contra a leishmaniose visceral (LV) canina pode contribuir para o controle da doença no homem. Visando o desenvolvimento de uma vacina contra LV canina, antígenos recombinantes de L. infantum foram selecionados em nosso laboratório pelo uso de uma mistura de soros de seres humanos ou cães naturalmente infectados pela L. infantum. Alguns destes antígenos foram testados em diversos protocolos de imunização, incluindo uso de diferentes adjuvantes, em camundongos ou cães. A imunização de camundongos ou cães com um dos antígenos recombinantes (rLci2B) usado isoladamente ou em associação com saponina induziu resposta imune Th2 ou Th1/Th2, respectivamente, não protetoras contra a infecção experimental. Com a determinação da sequência deduzida de aminoácidos notou-se que a maioria dos antígenos selecionados apresenta um segmento com sequência de aminoácidos única (domínio não repetitivo) e segmentos com sequência de aminoácidos com motivos repetitivos (domínios repetitivos). Possivelmente a incapacidade dos antígenos recombinantes de induzir uma resposta imune predominantemente Th1, protetora contra a LV, seria por conta da presença de domínios repetitivos, que favorecem a apresentação antigênica por linfócitos B e, consequentemente, estimulam uma resposta imune Th2. Para avaliar o direcionamento da resposta imune pelos dois tipos de domínio, novas construções de DNA foram concebidas de modo a codificar apenas domínio(s) não repetitivo(s) ou domínio(s) não repetitivo(s) e domínios repetitivos. OBJETIVOS: Produzir quatro proteínas recombinantes com domínios não repetitivos (rLci2-NT-CT, rLci3-NT-CT, rLci10-NT e rLci12-NT-CT) e avaliar a capacidade desses polipeptídios de induzir resposta imune celular in vitro em cães assintomáticos inoculados por via dérmica com L. infantum. MATERIAIS E MÉTODOS: Foram realizadas: a) a subclonagem de construções de DNA (Lci3-NT-CT, Lci10-NT e Lci12- NT-CT) em um plasmídeo apropriado para expressão em Escherichia coli, b) a determinação de condições apropriadas para produção das proteínas recombinantes (rLci2-NT-5R-CT, rLci2-NT-CT, rLci3-NT-2R-CT, rLci3-NT-CT, rLci10-NT-2R e rLci10- NT) c) a purificação das proteínas recombinantes por cromatografia de afinidade e d) avaliação da capacidade dos polipeptídios de induzir estimulação de células mononucleares sangue periférico (PBMC) de cães assintomáticos inoculados por via dérmica com L. infantum. RESULTADOS: Três (rLci2-NT-CT, rLci2-NT-5R-CT, rLci3- NT-CT, rLci3-NT-2R-CT, rLci10-NT e rLci10-NT-2R) dos quatro pares de polipeptídios recombinantes foram expressos, produzidos e purificados. Três antígenos recombinantes (rLci2-NT-5R-CT, rLci2-NT-CT e rLci3-NT-2R-CT) promoveram a linfoproliferação in vitro utilizando PBMC de cães assintomáticos inoculados por via dérmica com L. infantum CONCLUSÕES: Três das seis proteínas produzidas induziram a linfoproliferação, sendo a maior linfoproliferação encontrada para PBMC estimulado com a proteína sem domínios repetitivos (rLci2-NT-CT). Avaliações adicionais são necessárias para comprovar a utilidade destas moléculas em formulação de vacina contra leishmaniose visceral canina. / An effective vaccine against visceral leishmaniasis (VL) dog can help to control the disease in man. Aiming at development of a vaccine against canine VL, recombinant antigens of L. infantum were selected in our laboratory by using a mixture of sera from humans or dogs naturally infected with L. infantum. Some of these antigens were tested in different immunization protocols, including use of different adjuvants in mice or dogs. The immunization of mice or dogs with a recombinant antigens (rLci2B) used alone or in combination with saponin induced Th2 response or Th1 / Th2, respectively, did not protective against experimental infection. With the determination of the deduced amino acid sequence it was noted that most of the antigens selected segment has a unique amino acid sequence (non-repetitive domain) and segments of amino acid sequence with repetitive motifs (repetitive domains). Possibly the inability of recombinant antigens to induce an immune response predominantly Th1 protective against LV would be due to the presence of repetitive domains that promote antigen presentation by B cells and thus stimulate an immune response Th2. To assess the direction of the immune response by two types of domain, new DNA constructs were designed to encode only the domain (s) not repetitive (s) or domain (s) not repetitive (s) and repetitive fields. MATERIALS AND METHODS: Were performed: a) subcloning DNA constructs (rLci3-NT-CT, rLci10-NT and rLci12-NT-CT) into a suitable plasmid for expression in Escherichia coli, b) determining the appropriate conditions for the production of proteins recombinant (rLci2- NT-5R-CT, rLci2-NT-CT, rLci3-NT-2R-CT, rLci3-NT-CT, rLci10-NT-2R and rLci10-NT) c) purification of recombinant proteins by chromatography affinity d) evaluating the ability of polypeptides rLci2-NT-CT, rLci3-NT-CT and rLci10-NT to induce stimulation of peripheral blood mononuclear cells (PBMC) from healthy dogs inoculated dermal with L. infantum. RESULTS: Three (rLci2-NT-CT, rLci2-NT-5R-CT, rLci3-NT-CT, rLci3-NT-2R-CT, rLci10- NT and rLci10-NT-2R) of the four pairs of recombinant polypeptides are expressed, produced and purified. Three recombinant antigens (rLci2-NT-5R-CT, rLci2-NT-CT and rLci3-NT-2R-CT) promoted lymphocyte proliferation in vitro using asymptomatic dogs PBMC inoculated dermal L. infantum CONCLUSIONS: Three of the six proteins produced induced lymphoproliferation, most lymphoproliferation was found to PBMC stimulated with the protein without repetitive domains (rLci2-NT-CT). Additional evaluations are necessary to confirm the utility of these molecules against canine visceral leishmaniasis vaccine formulation

The overarching objective of this thesis was the development of an improved expression cell line for recombinant proteins, in which a transgene of interest can be inserted into a highly active gene locus using recombinase mediated cassette exchange. Random integration of transgenic DNA is a common route to achieve transgene expression. However, randomly integrated transgenes are susceptible to gene silencing over time, and do not show stable expression for extended periods in culture. Furthermore, every new cell clone generated requires regulatory approval. The improvement of expression strategies may significantly reduce the time required to bring novel recombinant protein products to the market. In order to identify a suitable expression locus for the integration of transgene expression cassettes, total protein samples were derived from the production cell lines HEK293 and CHO. Highly expressed proteins were isolated via 2D PAGE, and identified via peptide mass fingerprinting. Their promoter regions were then validated to express a recombinant transgene in HEK293 cells. The long term stability of these promoter regions was also assessed. Direct gene targeting of the highly active gene loci may or may not be possible in typical producer cell lines. Targeting of a murine homologue to these highly expressed CHO/HEK293 loci may be more efficient in a murine stem cell line. The transfer of a modified allele from HM1 murine embryonic stem cells, into a somatic cell line (HC11) was demonstrated in this thesis. These validated methods were then explored for the generation of viable HM1-HEK293 and HM1-CHO fusion hybrids. For these experiments, a fluorescence based fusion assay was generated, validated and used for in-situ monitoring of the cell fusion process. The random integration of transgenic DNA into mammalian genomes typically results in a highly unpredictable integration architecture. RMCE at such loci would be inefficient. However, a highly efficient RMCE reaction at (rare) single copy transgene integrations, may be possible under the correct conditions. RMCE at randomly integrated loci could therefore be more beneficial (for transgene expression) than random integration alone. This thesis explores this concept with the use of a randomly integrated RMCE platform, and subsequent selection of cell lines post RMCE attempts at these loci CRISPR/Cas9 technology was also applied to a highly expressed locus in HEK293 cells. A framework for successful direction of double strand breaks to a defined locus is demonstrated in this work. The methods used to achieve this can therefore be built upon for the homologous recombination of a transgenic cassette, into a highly expressed locus in HEK293 cells. Monoclonal antibodies have dominated the biologics market for over two decades, and mammalian expression systems are well suited to their production. The work in this thesis attempts to raise and verify antibody molecules against a potential tumour marker using hybridoma and phage display technologies.

Elastin like polypeptides (ELPs) are a class of naturally derived biomaterials that are non-immunogenic, genetically encodable, and biocompatible making them ideal for a variety of biomedical applications, ranging from drug delivery to tissue engineering. Also, ELPs undergo temperature-mediated inverse phase transitioning, which allows them to be purified in a relatively simple manner from bacterial expression hosts. Being able to genetically encode ELPs allows for the incorporation of bioactive peptides and functionalization of ELPs. This work utilizes ELPs for regenerative medicine and drug delivery. The goal of the first study was to synthesize a biologically active epidermal growth factor-ELP (EGF-ELP) fusion protein that could aid in the treatment of chronic wounds. EGF plays a crucial role in wound healing by inducing epithelial cell proliferation and migration, and fibroblast proliferation. The use of exogenous EGF has seen success in the treatment of acute wounds, but has seen relatively minimal success in chronic wounds because the method of delivery does not protect exogenous EGF from degradation, or prevent it from diffusing away from the application site. We created an EGF-ELP fusion protein to combat these issues. As demonstrated through the proliferation of human skin fibroblasts in vitro, the EGF-ELP may be able to aid in the treatment of chronic wounds. Furthermore, the ability of the EGF-ELP to self-assemble near physiological temperatures could allow for the formation of drug depots at the wound site and minimize diffusion, increasing the bioavailability of EGF and enhancing tissue regeneration. The objective of the second study was to create an injectable hydrogel platform that does not require conjugation of functional moieties for crosslinking or biological activity. Hydrogels are three-dimensional polymer networks that are able to absorb water and biological fluids without dissolving. Their high water content gives them physical properties similar to soft tissues, making them useful as scaffolds for cell migration and drug delivery vehicles. Injectable hydrogels that crosslink in situ are particularly useful because they can form to the shape of the defect, providing a near perfect fit. However, many hydrogel platforms cannot be crosslinked in situ because cytotoxic crosslinking reagents are required. Additionally, hydrogels typically require the chemical conjugation of crosslinking domains and bioactive peptides to the polymer backbone, adding more steps and time required for hydrogel production. We devised an injectable hydrogel platform that can be synthesized in a single step using photoreactive ELPs as the polymer backbone. Leucine auxotrophic Eshcherichia coli expressed ELPs containing photoleucine, a leucine analog and photoreactive diazirine crosslinker, which is substituted for leucine periodically throughout the ELP sequence. Upon exposure to ultraviolet radiation (~370 nm), photoleucine is able to form covalent crosslinks with amino acid side chains, forming a polymer network for hydrogel formation. Additionally, recombinant growth factors and morphogens can be encoded into the ELP sequence providing a simple method of hydrogel functionalization for regenerative medicine applications. The potential for this platform was demonstrated through in vivo crosslinking of photoreactive ELPs in the expression hosts. Though the production of the photoreactive ELP was not as forthright as originally assumed. The substitution of noncanonical amino acids typically requires the auxotrophic expression hosts to be starved of the amino acid that they are auxotrophic for. A noncanonical analog of said amino acid can then be supplemented into expression media, maximizing incorporation. In this investigation, it was found the addition of photoleucine alone inhibited photoreactive ELP expression. ELP expression only occurred in the presence of photoleucine if valine or leucine was also present in the media. Furthermore, valine was found to aid the production of ELPs as much as leucine. It was postulated the bacterial translational machinery might need to be altered for optimal ELP expression.

S. aureus é um patógeno que sempre surpreende equipes médicas quanto à sua capacidade de resistir em curto espaço de tempo às mais novas drogas lançadas no mercado. Algumas alterações genéticas que poderiam causar a emergência de VISA foram previamente identificadas, dentre elas uma mutação no sistema de dois componentes GraRS. As proteínas GraR e GraR*, esta última com a mutação que levaria ao fenótipo de VISA, foram estudadas com o intuito de resolver a estrutura tridimensional de ambas e determinar seu papel no processo de resistência à vancomicina. Ensaios de clonagem, expressão, purificação das proteínas e dicroísmo circular foram realizados, porém não houve sucesso na cristalização. No estudo epidemiológico, PFGE dividiu os 36 isolados de MRSA (25 de infecção e 11 de colonização) de um hospital mineiro em 8 pulsotipos. Análise do MLST e SCCmec do pulsotipo A (58,3% das amostras) mostrou que os isolados pertenciam ao CC5 (ST5 e ST105) e continham SCCmecII. Todos os 3 isolados ST239 continham SCCmecIII, sendo relacionados ao BEC. A maioria dos isolados ST5 continha SCCmecII como o Clone NY/J. Observou-se 24% de resistência à tigeciclina nos isolados de sítios de infecção. Dois isolados foram sensíveis à daptomicina depois de 24 horas de incubação mas resistentes após 48 horas. Os resistentes à tigeciclina eram todos ST105-SCCmecII, e de 5 pacientes diferentes. Os resistentes à daptomicina eram ST5-SCCmecII, de pacientes diferentes, e apresentaram algumas mutações no gene rpoB. Uma mudança na linhagem prevalente nos hospitais brasileiros tem sido reportada e, de fato, BEC não era prevalente neste hospital em 2009. ST5-SCCmecII e ST105-SCCmecII foram prevalentes, e ainda o último apresenta o agravante da resistência à tigeciclina quando esta droga ainda não era utilizada no hospital. Não houve disseminação de apenas um pulsotipo, sugerindo que essas linhagens sejam endêmicas ao hospital. O conhecimento do perfil das linhagens locais auxilia na adequação do tratamento empírico dado aos pacientes, além de demonstrar que é preciso ter cuidado ao se administrar novas drogas indiscriminadamente para evitar seleção de clones mais resistentes. / S. aureus is a pathogen that always surprises the medical staff regarding its capability to resist to the newest drugs marketed, in a short period of time. Some genetic changes which might cause the emergence of VISA were previously identified, among them a mutation on the twocomponent system GraRS. The proteins GraR and GraR*, the last one with the mutation that would lead to the VISA phenotype, were studied aiming to solve the tridimensional structure of both and determine their role on the process of vancomycin resistance. Cloning, expression, protein purification and circular dichroism experiments were performed, however, the crystallization was not successful. In the epidemiological study PFGE distributed the 36 isolates (25 from infection sites and 11 from colonization) from a hospital in Minas Gerais in 8 pulsotypes. Analysis of MLST and SCCmec of pulsotype A (58.3% of all samples) showed that the isolates belonged to CC5 (ST5 and ST105) and harbored SCCmecII. All three ST239 harbored SCCmecIII, being related to Brazilian Endemic Clone (BEC). Most ST5 isolates harbored SCCmecII as the NY/J clone. It was observed 24% of resistance to tigecycline on the infection sites isolates. On microdilution, 2 isolates were susceptible to daptomycin after 24 hours of incubation, but resistant after 48 hours. Tigecycline-resistants were all ST105-SCCmecII and were isolated from 5 different patients. Daptomycin-resistants were ST5-SCCmecII, from different patients, and they presented some mutations on the gene rpoB. A change in the prevalent lineage of the Brazilian hospitals has been reported and, in fact, the clone BEC was not prevalent in this hospital in 2009. ST5-SCCmecII and ST105-SCCmecII were prevalent, and also, the last one presents the aggravating factor of tigecycline resistance when this drug was not used in the hospital. However, there was no dissemination of only one pulsotype, suggesting these lineages to be endemic to the hospital. Knowledge of the profile of the local lineages helps on the adequation of empiric treatment given to the patients, besides demonstrating that care is needed on administering new drugs indiscriminately to avoid selection of the more resistant clones.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-11-28T12:32:36Z No. of bitstreams: 2 Dissertação - Emerth Mayra Hungria Pinto - 2013.pdf: 1722909 bytes, checksum: e6a82b8a35ca4753b2bb8ca10dd21280 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-11-28T13:14:01Z (GMT) No. of bitstreams: 2 Dissertação - Emerth Mayra Hungria Pinto - 2013.pdf: 1722909 bytes, checksum: e6a82b8a35ca4753b2bb8ca10dd21280 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-11-28T13:14:01Z (GMT). No. of bitstreams: 2 Dissertação - Emerth Mayra Hungria Pinto - 2013.pdf: 1722909 bytes, checksum: e6a82b8a35ca4753b2bb8ca10dd21280 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-02-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The development of laboratory tests applicable for the diagnosis/classification of the different clinical forms of leprosy is considered a research priority. The completion of Mycobacterium leprae genome together with new gene cloning/expression techniques and new bioinformatic tools have promoted the production and availability of new M. leprae recombinant proteins for immunologic assessment. Goal: This study assessed the serologic reactivity to M. leprae recombinant proteins among leprosy patients and controls from two hiperendemic regions in Brazil: “Rondonópolis/MT” and “Vila do Prata/Igarapé-Açú/PA” (former leprosy colony). Material and Methods: IgG antibodies to M. leprae recombinant proteins (92f, 46f, LID-1, ML0405 e ML1213; 2 g/ml) and IgM antibodies for the PGL-I synthetic trissacaride (NT-P-BSA; 0.01 g/ml) were detected by ELISA. The following study groups were included (n=847): newly diagnosed untreated leprosy patients (paucibacillary- PB and multibacillary- MB), household contacts of MB patients (HHC), healthy endemic controls (EC) and former MB that concluded leprosy multidrug therapy (MDT) (post MDT). Results: Among participants from Rondonópolis/MT (n=764), the seropositivity of MB patients (n=58) was 59% for 92f, 81% for 46f, 89% for LID-1, 84% for ML0405; 83% were anti-PGL-I positives. Among 10 anti PGL-I negative MB patients, 5 recognized LID-1 e 4 had IgG antibodies to 92f, 46f and ML0405. Among PB leprosy patients (n=93) seropositivity rates to M. leprae recombinant proteins ranged from 5-16%; 8% were anti PGL-I positives. In the HHC (n=192) and in the EC groups (n=282) low reactivity rates were detected ranging from 3-7%. For the post MDT group, positivity rates ranged from 17-45%. Among participants from “Vila do Prata” 3-8% of HHC were seropositives for recombinant proteins; 11% were anti- PGL-I positives. In the post MDT group from “Vila do Prata” (n=47) 33-50% were seropositives for recombinant proteins. Conclusion: High positivity rates to M. leprae- 46f, ML0405 e LID-1 were detected among MB leprosy patients with different genetic/ethnic profiles from distinct geographical regions. These results indicate that the development of a serologic test for leprosy employing both M. leprae recombinant proteins and PGL-I can potentially detect the majority of MB patients from different hiperendemic regions. The use of this diagnostic tool by the public health system can contribute for the early diagnosis and treatment of MB disease which are crucial for the control/elimination of leprosy in Brazil. / O desenvolvimento de testes laboratoriais para o diagnóstico/classificação das diferentes formas clínicas da hanseníase é tema prioritário em pesquisa. O sequenciamento completo do genoma do Mycobacterium leprae, avanços em técnicas de clonagem e expressão gênica e novas ferramentas de bioinformática promoveram a produção e a disponibilidade de novas proteínas recombinantes para avaliação imunológica. Objetivo: O objetivo deste estudo foi avaliar reatividade sorológica a proteínas recombinantes do M. leprae em pacientes com hanseníase e controles de duas áreas hiperendêmicas para hanseníase do Brasil: Rondonópolis/MT e Vila do Prata/Igarapé-Açú/PA. Materiais e Métodos: A presença de anticorpos IgG para as proteínas recombinantes do M. leprae (92f, 46f, LID-1, ML0405 e ML1213; 2 g/ml) e IgM para o trissacarídeo sintético do PGL-I (NT-P-BSA; 0.01 g/ml) foi avaliada por ELISA. Os grupos de estudo incluíram (n=847): pacientes recém diagnosticados com hanseníase (paucibacilar- PB e multibacilar- MB) virgens de tratamento, contatos domiciliares de MB (CD), controles saudáveis de área endêmica (CS) e casos antigos de hanseníase MB que concluíram a multidrogaterapia (MDT) (pós MDT). Resultados: Entre os participantes de Rondonópolis/MT (n=764), a soropositividade de pacientes MB (n=58) foi de 59% para 92f, 81% para 46f, 89% para LID-1, 84% para ML0405; 83% apresentaram IgM para PGL-I. Dentre 10 pacientes MB soronegativos para PGL-I, 5 reagiram com LID-1 e 4 reconheceram 92f, 46f e ML0405. Entre os pacientes PB (n=93) as taxas de sororeatividade para as proteínas recombinantes variaram de 5-16% e 8% apresentaram sorologia anti- PGL-I positiva. Nos CD (n=192) e CS (n=282) as taxas de reatividade variaram de 3-7%. No grupo pós MDT as taxas de positividade variaram de 17-45%. Na Vila do Prata a taxa de positividade para as proteínas recombinantes nos CD (n=36) variou de 3-8% e 11% dos CD apresentaram anticorpos anti-PGL-I. Entre os indivíduos pós MDT (n=47) as taxas de reatividades variaram de 33-50%. Conclusões: Taxas de positividade acima de 80% foram detectadas para proteínas recombinantes do M. leprae- 46f, ML0405 e LID-1 em pacientes com hanseníase MB com diferentes características genéticas/ étnicas de distintas regiões geográficas. Estes resultados indicam que o desenvolvimento de um teste sorológico para hanseníase que associe proteínas recombinantes e PGL-I tem o potencial de detectar a grande maioria dos pacientes MB provenientes de diferentes regiões hiperendêmicas. O uso deste teste pelo sistema de saúde pública poderá contribuir para o diagnóstico e tratamento precoces da hanseníase MB que são cruciais para o controle/eliminação da doença no Brasil.

A glicoproteína gp120 do HIV apresenta domínios conservados, dentre os quais está o C4. Este domínio está envolvido no reconhecimento da molécula CD4 na superfície das células alvo, e anticorpos dirigidos contra ele neutralizam o vírus. Em trabalho anterior, este domínio foi introduzido dentro da região globular da proteína fibra do adenovírus. Como a fibra adenoviral é estimulante do sistema imune, decidimos testar a capacidade de indução de anticorpos anti-C4 por essa proteína modificada. Assim, foram construídos vetores plasmídicos e adenovirais portando o gene da região globular da fibra adenoviral contendo o domínio C4, que usados na imunização de camundongos induziram anticorpos capazes de reconhecer a proteína gp120. Também construímos um vetor baculoviral expressando essa proteína híbrida, que purificada por HPLC, foi utilizada para imunizar camundongos que também produziram anticorpos capazes de reconhecer a proteína gp120. Nossos dados sugerem que a região globular da fibra adenoviral é uma boa plataforma para a exposição de epítopos de imunização. / HIV glycoprotein gp120 has conserved domains, one of them being the C4 domain. This region is involved in the recognition of the CD4 marker in target cells and antibodies that recognize this domain can block HIV infection. Previously, the C4 domain was introduced in the adenovirus fiber knob. As the adenovirus fiber stimulates de immune system, we decided to test the production of anti-C4 antibodies by this hybrid protein. We constructed plasmid and adenovirus vectors carrying the fiber knob modified with the C4 domain. Immunization of mice with these vectors showed the production of specific antibodies that recognized de gp120 glycoprotein. Also, we constructed a baculovirus vector expressing the hybrid protein, which was purified by HPLC. Mice immunized with this protein also produced antibodies capable of recognizing gp120. Our data suggest that the fiber knob is a good carrier protein for epitope immunization.

A dengue é uma doença infecciosa com grandes taxas de morbimortalidade, causada pelo vírus da dengue (DENV). Segundo a Organização Mundial de Saúde, cerca de 50 a 100 milhões de pessoas são infectadas anualmente em mais de 100 países tropicais e subtropicais de todos os continentes. O espectro clínico da infecção pelo DENV pode incluir formas assintomáticas ou sintomaticas que variam desde uma febre indeterminada e autolimitada, passando pela febre clássica da dengue (FD) até quadros graves denominados febre hemorrágica da dengue/síndrome do choque da dengue (FHD/SCD). Recentemente, ocorreu um dramático aumento do número de casos de FHD/SCD nas Américas, e este aumento coincidiu com a introdução do dengue sorotipo 3, genótipo III. No presente trabalho, objetivou-se a clonagem e a expressão das proteínas NS1 e NS3 do vírus da dengue tipo 3. As proteínas NS1 e NS3 do DENV-3 foram clonadas e expressas com sucesso em sistema procarioto. A amplificação dos genes das proteínas NS1 e NS3 foi realizada por RT-PCR, o qual gerou amplicons de cerca de 1050 e 1850 pb, respectivamente. Em seguida, os genes foram clonados por inserção dos amplicons no vetor plasmidial pCR-XL. Os genes de NS1 e NS3 foram subclonados no vetor de expressão pQE-30 através de sítios de restrição para as enzimas BamHI e HindIII. A expressão proteica foi obtida em sistema procarioto utilizando a cepa BL21(DE3) de E. coli, resultando em proteínas de 45 e 70 kDa as quais foram confirmadas por análises em Western blot utilizando como anticorpo primário fluido ascítico imune de camundongos e soro de pacientes com dengue. Estas proteínas virais podem ser utilizadas para estudos relacionados à patogênese, replicação e mecanismos de escape do sistema imune do DENV, além disso, podem ser potencias antígenos em métodos de diagnóstico. / Dengue is an infectious disease with high morbidity and mortality rates caused by dengue virus (DENV). According to the World Health Organization, about 50 to 100 million people are infected annually in more than 100 tropical and subtropical countries from all continents. The clinical spectrum of DENV infection can includes asymptomatic or symptomatic forms ranging from undetermined and self-limited fever, through dengue fever (DF) to severe disease called dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Recently, there has been a dramatic increase in the number of cases of DHF/DSS in the Americas, and this increase coincided with the introduction of dengue virus type 3 (DENV-3), genotype III. The present study aimed to clone and express NS1 and NS3 proteins of DENV-3. The NS1 and NS3 proteins of DENV-3 was successfully cloned and expressed in a prokaryotic system. Amplification of NS1 and NS3 genes was carried out by RT-PCR, which yielded amplicons of approximately 1050 and 1850 bp, respectively. Then, the genes were cloned by inserting the amplicons into the plasmid vector pCR-XL. NS1 and NS3 genes were subcloned into the expression vector pQE-30 through the restriction sites for BamHI and HindIII enzymes. The protein expression was obtained in a prokaryotic system using the strain BL21 (DE3) of E. coli, resulting in 45 and 70 kDa proteins, which were confirmed by Western blot analysis using immune mouse ascitic fluid and serum of patients with dengue as primary antibody. These viral proteins can be used to study the pathogenesis, mechanisms of replication and immune escape of DENV, moreover, can be potential antigens in diagnostic methods.

Sílicas mesoporosas, como a SBA-15, constituem-se de partículas de óxido de silício que, devido às suas propriedades físico-químicas apresentam potencial adjuvante. Para entender os mecanismos de ação da SBA-15, camundongos foram imunizados, pelas vias oral e/ou subcutânea (s.c.), com a proteína recombinante HBsAg ou ovalbumina (OVA) e apresentaram aumento significativo nos títulos de anticorpos específicos após imunização s.c. com ambos os antígenos em sílica; entretanto, somente a administração de HBsAg: SBA-15 induziu a produção de anticorpos pela via oral. O efeito da sílica na ativação de células dendríticas foi verificado após incubação com diferentes concentrações de SBA-15, indicando aumento na produção de IL-6, na proliferação e produção de IFN-g por linfócitos T após a apresentação de OVA ou seus peptídeos. A resposta de linfócitos T in vivo mostrou aumento na resposta imune celular de animais que receberam a sílica, mas não indicou a indução da resposta de linfócitos T citotóxicos. Esses resultados confirmam o efeito adjuvante da SBA-15, principalmente para a resposta de anticorpos, e indicam que a sílica pode aumentar a disponibilidade dos antígenos às APC, auxiliando na apresentação antigênica. / Amorphous silicon oxide particles named SBA-15 are promising adjuvant vectors due to its physicochemical properties and the aim of this study is to explore how they might act in promoting immune responses. Mice were orally and/or subcutaneously (s.c) immunised with the recombinant protein HBsAg or ovalbumin (OVA), showing a significant increase in the antibody titers after s.c. immunisation with both antigens in silica; however, only the administration of HBsAg: SBA-15 induced antibody production after oral immunisations. The activation of dendritic cell by silica was assessed by pulsing those cells with different concentrations of the particles, suggesting the interference of SBA-15 in the production of IL -6 and in T cell proliferation as well as IFN-g production by T lymphocytes after presentation of this protein or its peptides in vitro SBA-15 was able to enhance T cell responses in vivo but did not allow OVA to induce specific cytotoxic T lymphocyte activity. These preliminary data confirm that SBA-15 acts as an adjuvant for antibody responses and suggest that its effects may reflect enhanced availability of antigen, rather than direct effects on antigen presenting cells such as DC.

Nos últimos anos, estudamos vários aspectos da resposta imune naturalmente adquirida em indivíduos de diferentes áreas endêmicas da Região Amazônica expostos à malária. Para isso, utilizamos proteínas recombinantes baseadas em antígenos de formas sanguíneas de P. vivax, os quais têm sido considerados candidatos à vacina contra a malária vivax. No presente trabalho, nossos estudos imunoepidemiológicos concentraram-se em 396 indivíduos de uma comunidade rural da Amazônia ocidental brasileira, localizada no Estado do Acre, com o objetivo de realizar um estudo transversal e longitudinal da resposta de anticorpos contra um painel de proteínas recombinantes derivadas de merozoítos de P. vivax (MSP119, AMA-1, MSP3α e MSP3β). Para isso, os soros desses indivíduos foram testados por ELISA quanto ao reconhecimento das quatro proteínas recombinantes. As proporções de indivíduos na linha de base com anticorpos IgG contra MSP119, AMA- 1, MSP3α e MSP3β foram de 59,8%, 50,0%, 23,5% e 26,5%, respectivamente. Dentre esses indivíduos, apenas 10,9% tinham infecções por P. vivax, P. falciparum ou malária mista. No grupo de infectados por P. vivax, a proporção de respondedores foi maior para as proteínas recombinantes MSP119 e AMA-1, atingindo 78,6%, em ambos os casos. A proporção de indivíduos com anticorpos para cada uma das proteínas associou-se com o maior tempo de exposição à malária, exceto para a MSP3β. Além disso, a positividade foi mais alta nas áreas de maior risco de transmissão. Não observamos associações relevantes entre o genótipo do antígeno Duffy dos indivíduos e presença de anticorpos para as proteínas estudadas. No estudo longitudinal, observamos um aumento da prevalência de respondedores durante a parasitemia patente, sendo de 81,9%, 80,9%, 31,9% e 48,9% para a MSP119, AMA-1, MSP3α e MSP3β, respectivamente. Em conclusão, nossos resultados confirmam a alta antigenicidade dessas proteínas, o que pode ser de grande importância para futuros ensaios clínicos na região. / In recent years we studied various aspects of the naturally acquired immune response in individuals from different endemic areas of the Amazon region exposed to malaria. For this purpose we used recombinant proteins based on P. vivax blood stage antigens considered candidates for a vaccine against vivax malaria. In the present study, we focused on 396 individuals from a rural community in western Brazilian Amazon located at the state of Acre. We conducted a transversal and longitudinal study of the antibody response to a panel of recombinant proteins representing P. vivax merozoites surface antigens (MSP119, AMA-1, MSP3α and MSP3β). The sera of these individuals were tested by ELISA for the recognition of the four antigens mentioned above. The proportions of individuals at the baseline with IgG antibodies to MSP119, AMA-1, MSP3α and MSP3β were 59.8%, 50.0%, 23.5% and 26.5%, respectively. Among these individuals, 10.9% had patent malaria infections with either P. vivax or P. falciparum or both. Among individuals with patent P. vivax infection, the frequency of responders was high for MSP119 and AMA-1, (78.6% in both cases). Except in the case of MSP3β, the proportion of individuals with antibodies to each protein correlated with the time of malaria exposure. Also, the positivity was higher in areas of higher transmission levels. No relevant association was found between the Duffy genotypes and presence of antibodies to the different antigen. In longitudinal study, we observed an increased prevalence of responders during patent parasitemia, 81.9% 80.9% 31.9% and 48.9% to MSP119, AMA-1, MSP3α and MSP3β, respectively. In conclusion, our results confirm the high antigenicity of these proteins, which can be of great importance for future clinical trials in the region.

Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished