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|Title:||Recombinant intracellular antibodies for molecular gene therapy of HIV-1 infection|
|Authors:||Silva, Frederico Nuno Castanheira Aires da, 1974-|
|Orientador:||Gonçalves, João, 1967-|
Barbas III, Carlos F.
Teses de doutoramento - 2008
|Abstract:||The spread of HIV-1 has been dramatic since the early eighties, when the virus was discovered as the causative agent of AIDS. In the absence of an effective vaccine against HIV, a worldwide search has been made in the past two decades to develop small-molecule inhibitors to target essential steps in the viral cycle. Over the recent years, gene therapy has been highly regarded as a new form of molecular medicine in treatment of HIV/AIDS, either asan alternative or as a complement to anti-retroviral chemotherapy. An intrabody consists of an antibody designed to be expressed intracellularly and directed to different subcellular compartments where they can exert their function more effectively. The binding of an intrabody to its molecular target has the potential to block, suppress, alter or even enhance the process mediated by that molecule. Within this context, intracellular antibodies (intrabodies) represent a new class of neutralizing molecules with potential use in gene therapy approaches. The HIV 1 integrase (IN) protein is currently the focus of an intense research effort to develop new anti-HIV-1 drugs. This enzyme catalyses the integration of HIV genome into the chromosome of the host cell, arguably the most insidious step in the infection process. In the first project of this thesis (Chapter 2), we explored the intracellular immunization approach by developing rabbit intrabodies against the HIV-1 IN protein. We immunized rabbits with HIV-1 IN and developed a combinatorial scFv library againstIN. We were able to identify 5 different scFv's antibodies with high binding activity and specificity to IN. These scFv's bound simultaneously to the catalytic and C-terminus domains of IN. In addition, these antibodies have the ability to inhibit the strand transfer processing. Intrabody-expressing cells, either in their cytoplasm or nuclear compartments, were highly resistant to HIV-1 infection. Importantly, when HIV-1 particles where produced in the presence of anti-IN scFv, the expression of intrabodies did not affect virion production significantly. However, it markedly reduced the infectivity of progeny virions due to the incorporation of anti-IN scFv into the viral particles. These findings provide proof-in-principle that rabbit anti- IN intrabodies can be designed to block early and late stages of HIV-1 replication. As a result, our intrabodies might be useful agents for "intracellular immunization"- and used as new tools to study the structure and function of HIV-1 IN due to their epitope binding characteristics. Another potential target for HIV-1 treatment is Vif. This viral protein overcomes the innate antiviral activity of a cytidine deaminase APOBEC3G that induces G to A hypermutation in the viral genome, resulting in enhancement of viral replication infectivity. We previously demonstrated that anti-Vif scFv and camelized VH intrabodies are an effective approach to inhibit this crucial step of the viral replication cycle. In the second project of this thesis (Chapter 3), we showed that the rabbit VL domain can also be very potentially used as an intrabody. Our results demonstrate that the anti- Vif VL single-domain preserve the antigen-binding activity and specificity in the absence of the parent VH domain. In addition, the VL single domain was highly expressed in microbial cell culture and show favourable biophysical properties. The expression of the VL intrabody in eukaryotic cells also showed that the rabbit VL was correctly folded as soluble protein in the reducing environment and could strongly neutralize HIV-1 infectivity. Therefore, the present study suggests that rabbit VL single-domains have also an enormous value as intracellular antigen recognition units. Lentiviral vectors are among the most efficient tools for gene delivery into mammalian cells. A major goal of lentiviral gene delivery systems is to develop vectors that can efficiently target specific cell types. In the last project of this thesis (Chapter 4), we attempted to generate viral particles for targeting gene delivery. To achieve this goal we have used CCR5-positive cells as the target for our strategy. We designed a novel Sindbis pseudotyped vector where the Sindbis E2 receptor binding envelope protein was modified to directly encode a scFv against the CCR5 chemokine receptor. Targeting into specific cells was mediated by the anti-CCR5 scFv display, and viral titers were close to 106 EGFP transduction units/ml. Our data demonstrate that the length of the peptide linker that connects the heavy chain and light chain of anti-CCR5 scFv significantly affects the efficiency of infection. Infection levels obtained with Sindbis envelope displaying a scFv with a longer linker was consistently higher than that with Sindbis envelope displaying a scFv with a short linker. The results presented show that chimeric scFv-Sindbis pseudotyped lentiviral vectors have the potential to become an efficient and broadly applicable approach for targeting gene delivery to specific cells. Furthermore, this strategy has the potential to become a powerful approach for targeting gene delivery in anti- HIV gene therapy due to the important role of CCR5 expression in disease progression.|
|Description:||Tese de doutoramento em Farmácia (Microbiologia), apresentada à Universidade de Lisboa através da Faculdade de Farmácia, 2008|
|Appears in Collections:||FF - Teses de Doutoramento|
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