Vectors derived from Human Immunodeficiency Virus type 1 (HIV-1) are being widely developed for gene therapy applications, principally because they are able to transduce both dividing and non-dividing cells and result in stable, long term gene expression. However, these vectors are difficult to produce in high titres and sufficient volumes for large scale experiments and clinical application. Therefore, an investigation into methods to improve the production of HIV-1 derived gene transfer vectors was undertaken. One factor that limits the production of recombinant virus is the amount of viral genomic RNA available for packaging into virions. Therefore, a transfer vector was modified with the aim of increasing the amount of genomic RNA produced. Substitution of the polyadenylation (pA) signal, mutation splice donor sites and removal of unnecessary sequences were all examined. pA signal readthrough was quantified to determine the effect of these modifications on the rate of pA signal readthrough. Insertional mutagenesis and vector mobilisation are recognised risk factors with all integrating vectors. Self inactivating (SIN) vectors, which contain a deletion of U3 sequences in the 3’ LTR, demonstrate a reduced rate of mobilisation. Transduction with these vectors results in a provirus containing no viral promoter elements, with transcription of the transgene being controlled from an internal promoter. However, LTR repair of SIN vectors occurs at an appreciable frequency. Therefore, the extent of this deletion was maximised and the effect on the frequency of the repair examined. The production of lentiviral gene therapy vectors by large-scale transient transfection is both time consuming and technically difficult. Therefore, methods to increase the scale of production without compromising virus titre were developed. This resulted in fewer transfections and less handling of the cells when making virus on a large scale (3-4 L). In order to process the virus on this scale in a single day (i.e. 8 hours), new concentration and purification methods were established. The protocol consisted of low speed centrifugation, 0.45 μm filtration, 750 kDa ultrafiltration, 0.8 μm filtration and ultracentrifugation. However, the use of ultracentrifugation means that this protocol is not amenable to further scale up. Therefore, the replacement of the ultracentrifugation step with anion exchange was investigated. A number of different resins and anion exchange devices were investigated, two of which show promise for large scale purification of HIV-1 derived gene transfer vectors. In an ideal world, HIV-1 derived gene transfer vectors would be produced using stable packaging cell lines engineered to produce the desired virus. However, previous attempts to produce such a cell line with the desired properties have had limited success and have generally used outdated helper systems. Therefore, in an attempt to combine the efficiency advantages of having a single helper plasmid with the safety advantages of expressing each protein separately, a single packaging construct that contained separate transcription units for each of the required proteins was produced. Transcription of cyotoxic proteins was controlled by inducible promoters. Initial results suggest that such a system is technically feasible but that further work is required to optimise the expression of helper functions. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309550 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2008

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-05-14T13:06:07Z No. of bitstreams: 2 Dissertação - Marcella Silva de Paula - 2018.pdf: 2802750 bytes, checksum: 99659e0beca35484a0765a68de6bfdd7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-05-14T13:08:05Z (GMT) No. of bitstreams: 2 Dissertação - Marcella Silva de Paula - 2018.pdf: 2802750 bytes, checksum: 99659e0beca35484a0765a68de6bfdd7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-14T13:08:05Z (GMT). No. of bitstreams: 2 Dissertação - Marcella Silva de Paula - 2018.pdf: 2802750 bytes, checksum: 99659e0beca35484a0765a68de6bfdd7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-11 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / INI have been available in Brazil since 2009, when the first INI, Raltegravir, became available for therapy of rescue of HIV + patients in therapeutic failure. In the year 2017, a second INI was introduced into ART of patients newly diagnosed with HIV-1, Dolutegravir, which had a higher genetic barrier and a single daily dose, replaced Efavirenz in the first line of treatment. However, despite the efficiency of INI, the emergence of viral variants resistant to these drugs is inevitable. For this reason, it is necessary to monitor resistance mutations to INI, which may lead to therapeutic failure, aiming at optimizing the therapeutic regimen and controlling HIV infection. The objective of this study was to evaluate the occurrence of INI mutations and the resistance profile in HIV + / AIDS patients in the city of Jataí/Goiás. The complete IN gene was sequenced from samples from INI-naive patients. Resistance mutations were identified by the Stanford-HIV and IAS-USA database. Viral subtypes were identified by phylogenetic analysis. Among the 52 samples analyzed, no primary mutation was identified. Two accessory mutations (T97A / G163K) were identified and these induce a low level of INI resistance. In total, 152 polymorphisms were identified. The most prevalent subtype was subtype B. Therefore, these data demonstrate that the IN region is still highly conserved, encouraging the use of INI in HIV-1 therapy, and assist in the mapping of HIV-1 genetic diversity in the Southwest region of Goiás. / Os INI estão disponíveis no Brasil desde 2009, quando o primeiro INI, Raltegravir, passou a ser disponibilizado para terapia de resgate de paciente HIV+ em falha terapêutica. No ano de 2017, umsegundo INI foi introduzido na TARV de pacientes recém diagnosticados com HIV-1, o Dolutegravir, que por possuir barreira genética mais elevada e dose diária única, substituiu o Efavirenz na primeira linha de tratamento. Porém, apesar da eficiência dos INI, a emergência de variantes virais resistentes a estes fármacos é inevitável. Por isto, é necessário o monitoramento das mutações de resistência aos INI, que podem levar falha terapêutica, visando a otimização do esquema terapêutico e controle da infecção pelo HIV. O objetivo deste estudo foi avaliar a ocorrência de mutações aos INI e o perfil de resistência em pacientes HIV+/AIDS do município de Jataí/Goiás. O gene completo da IN foi sequenciado a partir de amostras de pacientes virgens de INI. As mutações de resistência foram identificadas pelo banco de dados de Stanford-HIV e IAS-USA. Os subtipos virais foram identificados por análise filogenética. Entre as 52 amostras analisadas, nenhuma mutação primária foi identificada. Duas mutações acessórias (T97A/G163K) foram identificadas e estas induzem baixo nível de resistência à Raltegravir e Elvitegravir. No total, 152 polimorfismos foram identificados. O subtipo mais prevalente foi o subtipo B. Portanto, estes dados demonstram que a região da IN ainda é bastante conservada, encorajando o uso de INI na terapia contra o HIV-1 e auxiliam no mapeamento da diversidade genética do HIV-1 na região Sudoeste Goiano

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-12-22T10:28:32Z No. of bitstreams: 2 Dissertação - Cristhiane Dias Rodrigues Schmaltz - 2011.pdf: 9277998 bytes, checksum: bdfa8d45ce0965166b22acba02729a7a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-12-22T10:29:13Z (GMT) No. of bitstreams: 2 Dissertação - Cristhiane Dias Rodrigues Schmaltz - 2011.pdf: 9277998 bytes, checksum: bdfa8d45ce0965166b22acba02729a7a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-12-22T10:29:13Z (GMT). No. of bitstreams: 2 Dissertação - Cristhiane Dias Rodrigues Schmaltz - 2011.pdf: 9277998 bytes, checksum: bdfa8d45ce0965166b22acba02729a7a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2011-03-28 / AIDS is responsible for over 2 million deaths each year, but the introduction of highly active antiretroviral therapy in 1996 has improved both the survival and the quality of life of patients. In Brazil, the Ministry of Health has been offering antiretroviral treatment free of charge for the last 15 years. However poor adhesion to treatment and extended use of antiretroviral drugs favor the selection and transmission of drug resistant viruses. This study assessed the clinical, virologic and immunologic responses of 97 patients genotyped before therapy between 2007-2008 by systematic revisions in the medical files for a medium period of 2 years. The pre-therapy genotypic tests identified 8/97 patients with transmitted resistance, 5/8 were men who have sex with men. According to WHO criteria similar clinical stage of the disease was observed in patients with and without resistant viruses. Among patients with wild virus 15/89 abandoned therapy, 4/89 were considered lost to follow up because they’ve been transferred to other clinics and 4/89 died, 3 due to AIDS causes, resulting in 66 patients followed up. Among 8 patients with transmitted resistance, one death not related to AIDS and 2 gave up therapy, resulting in 5 patients followed up. Upon therapy, patients with transmitted resistance presented an increase in the median of CD4+ cell counts of 215% whereas a 225% increase was observed in patients harboring wild virus. In both groups around 70% of patients had undetectable viremia following ARV treatment. The most common opportunistic infection was oral moniliasis. In conclusion, a wide variety of clinical outcomes was observed during short term follow up of patients genotyped pre-therapy. These outcomes ranged from therapeutic success represented by increase in CD4+ cell counts/undetectable viremia to death related or not to AIDS causes. A significant number of patients abandoned therapy. During follow up, there was no statistically significant difference in the clinical, virological and immunological outcomes of patients harboring wild or resistant virus. Altogether these results highlight the importance of pre-therapy genotypic tests for the adequate choice of ARV treatment preventing therapeutic failure and its immunological and clinical consequences, such as opportunistic infections and infections not related to AIDS. Moreover, the adequate prescription of ARV treatment avoids more complex drug regimens necessary for salvage therapy, which come at higher costs for the Brazilian public health system. / A aids é responsável por cerca de 2 milhões de mortes por ano, entretanto a introdução da terapia altamente eficaz em 1996 promoveu melhora significativa da sobrevida e qualidade de vida dos pacientes. No Brasil o Ministério da Saúde oferece gratuitamente medicação antirretroviral (ARV) há 15 anos. Porém a baixa adesão e o uso difundido de ARVs por longo prazo podem promover a seleção e transmissão de cepas virais resistentes a drogas ARV. Este estudo analisou resposta clínica, virológica e imunológica de 97 pacientes submetidos a genotipagem pré-terapia ARV do estado de Goiás entre os anos de 2007-2008. Revisões sistemáticas dos prontuários médicos foram realizadas por um período médio de dois anos pós a genotipagem. Resultados da genotipagem pré-tratamento indicaram 8/97 pacientes com resistência transmitida, sendo 5/8 homens que fazem sexo com homens. Segundo critérios da OMS o estadiamento clínico inicial dos pacientes com e sem resistência transmitida foi semelhante. Entre os pacientes com vírus selvagem, 15/89 abandonaram tratamento, 4/89 foram transferidos, 4/89 foram a óbito, dos quais 3 relacionados a aids, resultando em 66 pacientes acompanhados. Entre os 8 pacientes com vírus resistentes, ocorreu um óbito não relacionado a aids e dois pacientes abandonaram a terapia, resultando no acompanhamento de 5 pacientes. A terapia ARV nos pacientes com resistência transmitida levou a incremento na mediana das contagens de células CD4 + de 215% versus incremento de 225% em pacientes com vírus selvagens. Sob TARV em torno de 70% dos pacientes de ambos grupos apresentou carga viral indetectável. A infecção oportunista mais importante foi a monilíase oral em ambos os grupos. Em conclusão uma grande diversidade de desfechos clínicos foi observada em curto prazo após genotipagem para resistência. Estes desfechos variaram de sucesso terapêutico representado por incremento nas células CD4 + / viremia indetectável, a óbito por causa relacionada ou não à aids. Um número significativo dos pacientes abandonou tratamento ARV. Durante o acompanhamento não foi observada diferença estatisticamente significativa na evolução clínica, virológica e imunológica de pacientes com vírus resistente ou selvagem. Estes resultados destacam a importância da genotipagem pré-tratamento para escolha da terapia adequada, prevenindo falha terapêutica e suas repercussões imunológicas e clínicas, incluindo a instalação de doenças oportunistas e não relacionadas ao HIV. Além disso, a prescrição do esquema ARV adequado reduz a necessidade de esquemas terapêuticos mais complexos para terapia de resgate e de maior custo financeiro para o sistema de saúde pública brasileiro.

MSc (Med), Faculty of Health Sciences, University ofthe Witwatersrand, 2007 / Background Mother-to-child transmission of HIV infection is the primary cause of paediatric HIV infections worldwide. High HIV infection rates in women of childbearing age (15-49 years) and efficiency of PMTCT have resulted in the high rate of HIV incidence and prevalence in children of sub-Saharan Africa. The stark contrast in the success of PMTCT interventions between the western countries and less developed countries indicates the need for further research to develop alternative, easier, and more effective population-based interventions. Methodology This was a retrospective cohort study of the medical records of approximately 300 HIV infected women enrolled in the Nevirapine Resistance study between May 2002 and February 2003. An assessment of the significance of changes in immunological parameters (CD4 counts, CD4 percentages, CD4/CD8 ratios) and HIV RNA from 3rd trimester to 6 weeks postpartum and causal associations vi between these changes and increased risk of PMTCT was then conducted using logistic regression models. Results Mothers with CD4 counts above 200cells/μL were approximately exhibited onethird the likelihood of transmitting HIV-1 to their infants than mothers with CD4 counts below 200 cells/μL [OR 0.35 (0.13, 0.95)]. High maternal HIV RNA levels demonstrated a stronger association with increased risk of PMTCT with women with postpartum viral loads greater than 100 000 copies/μL exhibiting ten times the likelihood [OR 10.15 (2.17-47.55)]. Statistically significant mean increases in CD4 and CD8 cell counts from 3rd trimester to postpartum were observed. Mean increases in CD4 and CD8 counts demonstrated no association with PMTCT. Conclusion CD4 cell counts and CD8 cell counts underwent statistically significant changes from 3rd trimester to postpartum. These changes seem not to represent any clinically significant change in maternal disease progression during this time period and were found not to be associated with PMTCT.