HIV-1 has claimed the lives of millions of people globally and continues to spread despite development of highly active antiretroviral therapy. In 2013, 2.1 million new infections occurred and over 35 million people were living with HIV-1 infection. A prophylactic HIV-1 vaccine that can prevent infection or reduce viremia and subsequent transmission will always be an important part of the solution to bring this epidemic under control. In this thesis, the first HIV-1 vaccine candidate to focus on conserved regions of the virus (HIVconsv) was assessed in a phase I clinical trial conducted in healthy HIV-1/2 negative volunteers in Oxford. The HIVconsv T-cell immunogen was delivered using three leading vaccine modalities (DNA (D), modified vaccinia virus Ankara (M) and chimpanzee adenovirus serotype 63 (C)), in several novel heterologous prime-boost regimens. The frequency of T cells elicited through HIVconsv vaccination in the CM and DDDCM regimens surpassed that of previous HIV-1 cell-mediated vaccines. A large proportion of these T cells produced multiple cytokines and proliferated in response to recall peptides. The breadth of T-cell responses were also greater than the non-efficacious STEP study vaccine, with an average of 10 T-cell epitopes per vaccine recipient recognised across CM and DDDCM regimens. In vitro HIV-1 control mediated by CD8⁺ T cells was demonstrated for all vaccinees receiving the CM regimen, mainly against clade A (U455) and clade B (IIIB) isolates. Two vaccinees, demonstrated superior control of 6/8 and 7/8 viruses from the panel. The CM regimen induced significantly higher magnitudes of viral inhibition compared to the DDDCM or DDDMC regimens, with this regimen showing potential to overcome the disadvantage for subjects of carrying non-protective HLA alleles. Investigation of T-cell specificities revealed that the frequencies of T cells specific for conserved Gag but more so Pol regions significantly correlated with in vitro virus control. Direct examination of peptide expanded T-cell lines showed that all Pol pool- and limited Gag pool-specific cell lines reduced HIV-1 replication in vitro. In most individuals, targeting multiple HIV-1 epitopes concomitantly resulted in higher levels of virus inhibition than targeting a single viral epitope and two T-cell specificities showed enhanced control of HIV-1; the first within Pol (TAFTIPSI) and second from Gag (TERQANFL). These data support further development of the conserved region strategy for T-cell vaccines against HIV-1.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-05-07T22:43:52Z No. of bitstreams: 1 126110038.pdf: 2182930 bytes, checksum: 1d0e56324e775d985abbaf5cea8f4038 (MD5) / Made available in DSpace on 2015-05-07T22:43:52Z (GMT). No. of bitstreams: 1 126110038.pdf: 2182930 bytes, checksum: 1d0e56324e775d985abbaf5cea8f4038 (MD5) Previous issue date: 2014-03-21 / As abelhas apresentam uma combinação de características individuais e ainda de cooperação animal não encontrada no restante do reino animal. São insetos sociais e participam da polinização de diversas plantas que fornecem alimento para o homem. No Brasil com a africanização das abelhas, essas tornaram-se altamente produtoras e enxameadoras, o que vem tornando o país uma potência na produção de mel e outros produtos originados da atividade apícola. Muitas doenças podem afetar as abelhas, dentre elas muitas causadas por vírus. Controlar as infecções virais é essencial para a manutenção ecológica das abelhas e da produção apícola. Ao mesmo tempo, existe a possibilidade de relacionar vírus que infectam humanos com os vírus que infectam as abelhas, visto que os tratamentos utilizados para os seres humanos poderiam ser utilizados em colméias e, ao mesmo tempo, utilizar as abelhas como modelo de estudo para o desenvolvimento de novos antivirais. Na busca por um ponto em comum analisamos filogeneticamente a protease 3C, que ocorre nos vírus da super-família Picorna-like, onde encontram-se os vírus que parasitam as abelhas. Essa protease tem a capacidade de clivar a poliproteína viral nas proteínas maduras do vírus e ainda causar a degradação proteolítica das proteínas do hospedeiro. Até hoje não foi encontrada uma forma de utilizar a protease 3C em estudos filogenéticos pois existe muita divergência das suas sequências entre os vírus. O objetivo dessa pesquisa foi identificar uma forma de analisar filogeneticamente a protease 3C. As sequências da protease 3C e da RdRp de 55 vírus foram coletadas do NCBI ( National Center of Biotechnology Information) e submetidas ao MEME ( Multiple Em for Motif Elicitation), onde foram obtidos quatro sítios conservados. Após foi realizada a análise filogenética dos sítios conservados por Máxima Verossimilhança e análise da distância entre os sítios por parcimônia e ainda foi construída uma árvore com base na RdRp. As árvores dos sítios 1 e 2 apresentaram uma melhor robustez estatística e agrupamento dos vírus. Essas regiões conservadas da protease 3C-Pro podem ser o início para estabelecermos uma relação entre as proteases dos picornavírus e vírus picorna-like na busca da compreensão do seu mecanismo de infecção viral e também uma alternativa de estudo para outras sequências com alta variabilidade. O uso dos domínios 1 e 2 proporcionou a árvore com maior robustez apresentada até o dia de hoje para esta proteína viral. / The bees have a combination of individual features and animal cooperation is not yet found in the rest of the animal kingdom . They are social insects and participate in the pollination of many plants that provide food for man . In Brazil with the africanization of bees , these have become highly producing and swarm , which is making the country a power in the production of honey and other products derived from beekeeping . Many diseases can affect bees , among them many caused by viruses . Control viral infections is essential for ecological maintenance of bees and beekeeping . At the same time , it is possible to relate viruses that infect humans and viruses that infect the bees , whereas the treatments for humans could be used in beehives and at the same time using the bees as a model for development of new antiviral agents. In the search for a common point analyzed phylogenetically 3C protease , which occurs in the superfamily Picorna -like, which are viruses that parasitize bees virus. This protease is capable of cleaving the polyprotein, the mature viral proteins and viruses also cause the proteolytic degradation of host proteins . Until today there a way to use the 3C protease was found in phylogenetic studies because there is much divergence of their sequences between virus.The objective of this research was to identify a way to analyze phylogenetically 3C protease . The 3C protease and RdRp sequences of 55 viruses were collected from the National Center for Biotechnology Information , submitted to MEME , where four conserved sites were obtained . Upon phylogenetic analysis of the conserved sites and by maximum likelihood analysis of the distance between sites by parsimony was performed and was still a tree constructed based on the RdRp . Trees of sites 1 and 2 had a better statistical robustness and clustering of virus. These conserved regions of the 3C protease - Pro may be the start to establish a relationship between the proteases of the picornavirus and Picorna-like viruses in the quest to understand the mechanism of viral infection and also an alternative study for other sequences with high variability . The use of domains 1 and 2 provided the tree with greater robustness displayed until today for this viral protein.