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Publication
Molecular analysis of HIV under the pressure of protease inhibitors
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| 5.45 MB | Adobe PDF |
Abstract(s)
The Human Immunodeficiency Virus is the causative agent that leads to AIDS. One of the
enzymes that compose the viral particle is an aspartic protease that cleaves polyproteins
during viral replication. This enzyme is necessary during the maturation process of the
virions, and that is a preferential target for the design of new drug therapies, such as the
protease inhibitors (PI). The aim of this thesis was to examine the effect of single amino
acid substitutions in HIV-2 PR both in terms of resistance to PIs and in terms of drug free
infectivity. For each mutant we have determined the level of its selective advantage relative
to wild type virus over a range of drug concentrations. We have also characterized
the HIV-2 proteases from patients under PI treatment genotipically and evaluated their
phenotipically resistance to four selected PI by a recombinant viral assay (RV A), allowed
us to study the mechanism of action of anti-HIV drugs. The results obtained from RV A
may reflect a different pattern of HIV-2 resistance when compared with HIV-1 and in
vitro studies can provide insight in what we expect in clinical trial. We have also developed
a new functional cell based assay (CBA) for testing resistance that allows monitoring
HIV-1 protease activity and susceptibility to PI in living cells without the need for
culture infectious HIV. The signal obtained correlates with the intracellular activity of the
protease and the optimized cleavage assay was used for testing current PIs to compare
the IC50 values with virologic assays. The advantage of CBA is that we can have results
within 48 hours, two times faster than a RV A, and can be used in reference laboratories
as a powerful tool for identifying novel PIs. A third approach was made by screening
an in vitro phage-display library of IgNAR variable domains against the target antigen
HIV-1 protease to develop a novel monoclonal antibody against this enzyme. We used a
single-chain antibody naïve library based in the variable domain of IgNAR cloned into
pComb3X phagemid. Forty-eight possible anti-protease clones were selected and Phage-
ELISA and ELISA tests showed the specific binders. The data obtained from screnning
library of IgNAR have proved that the obtained antibodies were functional against the
HIV-1 protease viral particle and that can be used in the future as a new gene therapy
strategy with several potentialities in infectious diseases therapeutics
Description
Tese dout. , Ciências Biotecnológicas, 2008, Universidade do Algarve
Keywords
Teses Biotecnologia Biologia molecular SIDA Anticorpos 577.2