Utilize este identificador para referenciar este registo: http://hdl.handle.net/10451/32315
Título: Characterization of HIV-2 susceptibility to protease and entry inhibitors and identification of envelope determinants of coreceptor usage, cell tropism and antibody neutralization
Autor: Martins, Andreia Rodrigues, 1983-
Orientador: Taveira, Nuno, 1964-
Pereira, J. Moniz, 1949-
Palavras-chave: Teses de doutoramento - 2018
Data de Defesa: 2018
Resumo: The main aim of this work was to characterize the susceptibility of HIV-2 to protease and entry inhibitors and to identify viral determinants of coreceptor usage, cellular tropism and antibody neutralization. The specific objectives were: 1) to determine the contribution of amino acids residues in the V3 loop involved in CCR5 and CXCR4 use, susceptibility to antibody neutralization and cellular tropism; 2) to develop a genotypic method for the prediction of HIV-2 coreceptor usage based on V3 loop; 3) to evaluate the antiviral activity of a new short-peptide fusion inhibitor in HIV-2 and 4) to characterize the evolution and diversity of protease (PR) in HIV-2 infected patients treated and untreated with protease inhibitors (PIs). In the first study (Chapter 3), site-directed mutagenesis was used to create amino acid substitutions in residues 18 and/or 29 and/or single deletions at positions 23 and 24 in V3 loop of pROD10, an infectious molecular clone of HIV-2ROD, the reference X4 isolate. Cellular assays demonstrated that: 1) conversion from X4 to R5 phenotype in HIV-2ROD10 requires H18L substitution and the deletion Δ(23,24); 2) H18L and H23Δ + Y24Δ mutants are more easily neutralized than HIV-2ROD and other mutated viruses by plasma from HIV-2 infected individuals; on the other hand, K29T substitution seems to contribute to increase resistance to neutralization; 3) K29T mutants acquire macrophage tropism without compromising replicative capacity in CD4+ T lymphocytes; 4) H18L + Δ(23,24) and (23,24) mutants gained the ability to replicate in macrophages albeit at the cost of some capacity to replicate in CD4+T cells. Structural analysis by homology modelling showed that: 1) H18L substitution disrupts the interaction of histidine with methionine at position 15 and with phenylalanine at position 20; 2) deletion of H23 and Y24 leads to the elimination of the parallel β sheets presented in the V3 loop and the loss of the aromatic system which can compromise the binding of cellular coreceptors or other molecules (e.g. antibodies); 3) K29T substitution reduces the charge of V3 and leads to the loss of the interactions with isoleucine at position 27. Collectively, these results demonstrated that V3 is an important determinant in HIV-2 coreceptor usage, susceptibility to antibody neutralization and replication capacity on CD4+ T cells and macrophages and that these phenotypic characteristics can be modulated by a single amino acid change in V3. These results support an important role for V3 in the pathogenesis of HIV-2 infection. In the second study (Chapter 4), a genotypic method was developed for the prediction of HIV-2 coreceptor usage from the V3 loop, similar to an existing tool created for HIV-1 (geno2pheno [coreceptor-hiv2]). The development and validation of this tool was based on a data set of 126 samples from HIV-2 infected patients, most of them from Portugal, with phenotypic coreceptor usage annotations. Predictive accuracy was also validated based on the V3 mutants produced and phenotypically characterized in the previous chapter. Overall, these findings indicated that geno2pheno [coreceptor-hiv2] can be a useful tool in clinical practice, allowing better management of HIV-2 infected patients eligible for maraviroc (MVC). In the third study (Chapter 5) a short-peptide named 2P23 was produced by combining a M-T hook structure, HIV-2 sequences and ‘salt-bridge’-based strategies. This peptide showed a potent antiviral activity against HIV-2 and HIV-1 isolates (mean 50% inhibitory concentration- IC50: 20.17 nM and 5.57 nM, respectively) and SIV (IC50: 1.8 nM for SIVpbj and 3.29 for SIV239). This new fusion inhibitor also demonstrated a strong activity against the V3 variants (Chapter 3) (IC50:15.38 nM), irrespectively of the coreceptor phenotype. Thus, 2P23 is an ideal candidate for further clinical development due to its broad antiviral activity against several HIV-2 isolates, with different coreceptor tropism. The last study (Chapter 6), involved the characterization of PR diversity and genotypic resistance to PIs of HIV-2 infected individuals living in Portugal and the evaluation of the impact of resistance mutations to PIs in treatment outcome eight years post-therapy. A high prevalence of PR mutations (e.g. I54M, I82F, L90M) associated to saquinavir (SQV), darunavir (DRV) and lopinavir (LPV) resistance, were detected in proviral DNA from these patients at baseline. Eight years after study entry, the genotypic analysis identified: 1) loss of resistance mutations in two patients, that were initially detected at baseline, presumably as a consequence of treatment interruption; 2) long term persistence of resistance mutations in one individual as a result of virologic and immunologic failure, which might raise concern about transmission of drug resistance in the future and 3) development of new resistance mutations in three patients due to previous treatment failures. The analysis of genetic diversity in PR showed an increase in this parameter in two treated patients, with undetectable viral loads and higher CD4+ T counts, comparing with the baseline. On the other hand, a reduction in PR genetic diversity was exhibited in three patients (two treated and one untreated), who presented detectable viral loads in at least one time point during the follow up. Due to small sample size it was not possible to investigate a potential relationship between PR genetic diversity and CD4+ T cell counts, presence of resistance mutations or/and treatment status. However, these results seem to indicate a persistent viral replication during long term highly active antiretroviral therapy (HAART), regardless of plasma viral load. The maintenance of viral replication can act as a source of new proviral quasispecies, resulting in the gradual substitution of the ancestral variants over time. Most importantly, we found two potential cases of transmitted drug resistance. However, due to the small sample size, additional studies with a higher number of patients are required to determine if primary drug resistance is a major problem in HIV-2 infected patients in Portugal. Our findings suggest that proviral DNA may be useful in resistance testing in HIV-2 patients with low or suppressed viremia and in untreated patients, and that early resistance analysis of these archived viruses may predict treatment response.
Descrição: Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2018
URI: http://hdl.handle.net/10451/32315
Designação: Doutoramento em Farmácia
Aparece nas colecções:FF - Teses de Doutoramento

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