Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2017-05-05T15:28:25Z No. of bitstreams: 1 Dissertação_ISC_Luciana Santos Cardoso.pdf: 4791824 bytes, checksum: adeea3b3d364d4f4df9afdcb3f7f0a53 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-05-23T13:28:10Z (GMT) No. of bitstreams: 1 Dissertação_ISC_Luciana Santos Cardoso.pdf: 4791824 bytes, checksum: adeea3b3d364d4f4df9afdcb3f7f0a53 (MD5) / Made available in DSpace on 2017-05-23T13:28:10Z (GMT). No. of bitstreams: 1 Dissertação_ISC_Luciana Santos Cardoso.pdf: 4791824 bytes, checksum: adeea3b3d364d4f4df9afdcb3f7f0a53 (MD5) / Estudos vêm demonstrando diminuição da reatividade aos testes cutâneos para alérgenos e menor gravidade da asma em indivíduos infectados por helmintos, principalmente Schistosoma mansoni. A inibição da resposta inflamatória na asma em indivíduos infectados pelo S. mansoni parece ser mediada pela IL-10, desde que tem sido observada maior produção desta citocina por células de asmáticos infectados pelo S. mansoni estimuladas com o antígeno 1 do Dermatophagoides pteronyssinus (Der p1) quando comparado a asmáticos não infectados. A IL-10 é capaz de inibir a produção de citocinas do tipo Th2 e a degranulação de mastócitos e liberação de mediadores inflamatórios, fatores envolvidos na patogênese das doenças alérgicas. O principal objetivo deste estudo foi avaliar a capacidade dos antígenos de S. mansoni Sm22.6, Sm14, PIII, P24 e Sm29 de estimular a produção de IL-10 in vitro, por células mononucleares de sangue periférico de asmáticos infectados e não infectados pelo S. mansoni. Foram também avaliadas as produções de IL-5, IL-13 e IFN-g. Foi adicionado Sulfato de Polimixina B às culturas estimuladas com os antígenos recombinantes para bloquear a ação da endotoxina em induzir a síntese de citocinas, desde que as proteínas recombinantes foram clonadas em Escherichia coli. As concentrações das citocinas foram medidas nos sobrenadantes das culturas de células utilizando-se a técnica ELISA sanduiche. Foi demonstrado que todos os antígenos de S. mansoni avaliados neste estudo induziram a produção de IL-10 por células de indivíduos infectados e de asmáticos não infectados. Nas culturas de 24 horas de células de asmáticos não infectados, os antígenos P24 e Sm 29 induziram as mais altas concentrações de IL-10 (828 ± 415 pg/mL e 891 ± 213 pg/mL,respectivamente). Em todos os grupos avaliados e para todos os antígenos usados, foi baixa a produção de IFN-g (valores em torno de 100 pg/mL) e os níveis de IL-5 foram abaixo do limite de detecção (15,6 pg/mL). A produção de IL- 13 induzida pelos antígenos Sm22.6, P24 e PIII foi avaliada no grupo de asmáticos não infectados, e foram observadas concentrações de 68 ± 60 pg/mL, 55 ± 49 pg/mL e 81 ± 67 pg/mL, para os respectivos antígenos. A adição dos antígenos de S. mansoni Sm22.6, P24 e PIII às culturas de asmáticos não infectados estimuladas com o Der p1 resultou em aumento na produção de IL-10. O fato dos antígenos de S. mansoni avaliados neste estudo terem induzido a produção de IL-10 e baixas concentrações de IL-5, IL-13 e IFN-g por células de asmáticos não infectados sugere que os mesmos pode ser futuramente utilizados como vacina para prevenir doenças alérgicas.

Made available in DSpace on 2016-06-02T20:21:32Z (GMT). No. of bitstreams: 1 DissPVC.pdf: 931304 bytes, checksum: 56aee4360d52be36ef0d88b8beb6b9b2 (MD5) Previous issue date: 2004-01-01 / Proteins from the inner core of HIV-1 are involved in crucial processes during the virus life cycle. p24 is the major capsid protein of HIV and is initially expressed as part of the gag polyprotein. The association of gag proteins to the cell inner-membrane surface initiates virus assembly and induces budding from the host cell membrane. Thus, p24 plays an active structural role both as part of the Gag protein and in its mature form. In this sense, we have chosen a region from C-terminal of p24, TLRAEQASQEVKNWMTETLLVQNA, (p24-3) which is part of the major region responsible for protein dimerization. The linear peptide, rp24-3, and its cyclic variant, rp24-3m, were produced by recombinant strategy in Escherichia coli. The gene fragments were obtained by the synthetic gene approach and inserted into pET 32a to produce fusion proteins in the soluble form. The expression products were purified by Ni-affinity chromatography followed by an enzymatic cleavage. The peptides where purified by reverse phase chromatography and their primary sequence and molecular masses where inferred by amino acid sequence analysis and mass spectrometry, respectively. The rp24-3 secondary structure was investigated by circular dichroism and steady state fluorescence, been structured differently in water and in buffer. Besides, its tryptophan is in a partially buried environment and the addition of methanol above 70% caused a highly increase in helical content. In conclusion, this work shows a suitable system for rp24-3 production, providing satisfactory amount for structural studies. / As proteínas do centro do HIV-1 estão envolvidas em processos cruciais durante o ciclo de vida viral. A p24 é a principal proteína do capsídeo do HIV e é inicialmente expressa como parte da poliproteína Gag. A associação das proteínas Gag na superfície da membrana interna da célula hospedeira dá início à montagem viral e desencadeia o processo de brotamento da membrana da célula hospedeira. Dessa forma, a p24 possui um importante papel estrutural tanto no contexto da Gag, como na sua forma madura. Nesse sentido, foi escolhido um peptídeo da região C-terminal da p24, TLRAEQASQEVKNWMTETLLVQNA, (p24-3) que compõe a região principal responsável pela dimerização da proteína p24. O peptídeo linear, rp24-3, e sua variante cíclica, rp24-3m, foram produzidos em Escherichia coli via estratégia recombinante. Os fragmentos gênicos foram obtidos por meio da montagem de genes sintéticos e foram inseridos no vetor pET 32a para a produção como proteínas de fusão na forma solúvel. Os produtos expressos foram purificados por cromatografia de afinidade em Ni e submetidos a uma clivagem enzimática. Os peptídeos foram então purificados por cromatografia em fase reversa e suas sequências primárias e massas moleculares foram inferidas por meio do sequenciamento de aminoácidos e análises por espectrometria de massa, respectivamente. A estrutura secundária do rp24-3 foi investigada por dicroísmo circular e fluorescência estática, mostrando-se estruturado diferentemente em água e em PBS. Além disso, o triptofano está em um ambiente parcialmente escondido. A adição de metanol acima de 70% causou um grande aumento no conteúdo de hélices. Concluíndo, este trabalho mostra um sistema viável para a produção do rp24-3, proporcionando quantidades necessárias para a realização de estudos estruturais.

<p>As of 2021, 38.4 million people worldwide are living with Human Immunodeficiency virus (HIV), with eastern and southern Africa having the highest prevalence. The efficacy of treatment is determined by identifying acute HIV infections (AHI) and prompting early antiretroviral therapy (ART) initiation to achieve viral suppression and reduce the risk of transmission. Existing rapid tests that detect host antibodies are affected by long seroconversions which allow the viruses to remain undetected until long after infection. On the contrary, highly sensitive nucleic acid amplification test (NAAT) based assays, serving as the gold standard for detection are restricted by their long turnaround time and high cost of implementation thus, restricting their use in low resource settings. Further, drug resistance cases and patient non-compliance to treatment may lead to HIV progression to aids; therefore, effective viral load monitoring is a critical component in the HIV care continuum. To address the gaps in viral load monitoring and early HIV detection, I propose to develop assays for handheld self-test platforms to detect low concentrations of HIV via two different approaches: 1) I will optimize an existing NAAT - based assay to semi-quantitatively detect HIV particles that were spiked in clinical samples and 2) I will Investigate the binding kinetics between HIV p24 antigen and Anti-HIV-1 p24 Antibody using the principle of Bio-layer Interferometry. Thus, I will lay the foundation for the development of a novel and highly sensitive p24 detection assay. Overall, this work will enable detection of ahi detection as well as support people living with HIV (PLHIV) management, all while remaining connected to healthcare and provider support. </p> <p><br></p>

These studies were aimed at investigating the potential application of nanoparticles engineered from oil-in-water microemulsion precursors for enhancing immune responses to HIV-1 Tat and Gag p24 proteins. Both of the HIV-1 proteins have been reported to be critical in the virus life cycle and are being evaluated in clinical trials as vaccine candidates. Anionic nanoparticles were prepared using emulsifying wax as the oil phase and Brij 78 and sodium dodecyl sulfate as the surfactants. The resulting nanoparticles were coated with Tat and were demonstrated to produce superior immune responses after administration to BALB/c mice compared to Tat adjuvanted with Alum. Similarly, cationic nanoparticles were prepared using emulsifying wax and Brij 78 and cetyl trimethyl ammonium bromide as the surfactants. The cationic nanoparticles were investigated for delivery of immunostimulatory adjuvants, namely three Toll-like receptor ligands, for obtaining synergistic enhancements in immune responses to a model antigen, Ovalbumin (OVA). In vitro and in vivo studies were carried out to elucidate possible mechanisms by which nanoparticles may result in enhancements in immune responses. In vitro studies were carried out to evaluate the uptake of nanoparticles into dendritic cells and to assess the release of pro-inflammatory cytokines from dendritic cells in the presence of nanoparicles. In vivo studies were carried out using a MHC class I restricted transgenic mouse model to investigate the potential for nanoparticles coated with OVA to enhance presentation of the protein to CD8+ T cells compared to OVA alone. Finally, the preparation of nanoparticles with a low amount of surface chelated nickel for high affinity binding to histidine-tagged (his-tag) proteins was investigated. It was hypothesized that this strengthened interaction of his-tag protein to the nickel chelated nanoparticles (Ni-NPs) would result in a greater uptake of antigen in vivo; therefore, enhanced immune responses compared to protein bound to anionic nanoparticles. In vivo evaluation of his-tag HIV-1 Gag p24 bound to Ni-NPs resulted in enhanced immune responses compared to protein either adjuvanted with Alum or coated on the surface of nanoparticles.

Objective: To establish stable transgenic non-toxic plants as a platform for plant-based vaccine production as well as potential oral delivery system of vaccine antigens for sexually transmitted infections (STIs). The concept is to immunize the mucosal immune system present in the gut-associated lymphoid tissues (GALT). HIV-1 p24 subtype C protein has been used as the main antigen model, in parallel with an engineered unique chimeric MOMP antigen from Chlamydia trachomatis serovar E. Methods: Chimeric MOMP and p24 vaccine antigens were successfully inserted into the nuclear genomes of Arabidopsis thaliana and Daucus carota via Agrobacterium-mediated gene transfer. The characteristics of the genetic inserts and corresponding mRNAs and recombinant proteins in planta were described using several methods, including northern, Southern, and western blotting, ELISA, and a commercial HIV Ag/Ab combination assay. Immunogenicity of the antigens was studied in mice models. Results: Transgenes of both plant species expressing p24 or chimeric MOMP were successfully generated. Additional HIV-1 vaccine antigen candidates were introduced and the genetic inserts have been confirmed in Arabidopsis thaliana. The Arabidopsis thaliana expressing p24 and chimeric MOMP were demonstrated to be stable over generations and antigenicity analyses showed that plant-derived HIV-1 p24 and chimeric MOMP retained immunological epitopes when they were expressed in planta. Oral administration of transgenic plant material generated a priming effect of the immune competent cells present in the GALT, shown by the presence of antigen-specific-IgG in mice sera after boosting. Mice immunized with plant-derived HIV-1 p24 antigen were also analyzed for antigen-specific faecal IgA as well as cellular immune responses. However, detectable levels of the two latter immune responses were not observed. The Chlamydia trachomatis chimeric MOMP antigen was further evaluated for its potential as a vaccine antigen candidate, with positive results indicating a more rapid clearance of the Chlamydia trachomatis infection post immunization. Conclusion: Stable non-toxic transgenic plants expressing either HIV-1 p24 or a novel  Chlamydia trachomatis chimeric MOMP antigens have successfully been developed. The two plant-produced STI vaccine antigens have in initial mice feeding studies provided important proof-of-concept for the oral vaccination approach. Now, immunization studies to expand, en-hance, and improve knowledge of the immune responses generated by the orally delivered transgenic plants are of high priority. / Kemi/biokemi