Impact of integrase polymorphisms and minor quasispecies in HIV-1 infected individuals naive or treated with strand-transfer integrase inhibitors: a refined analysis by cloning and 454-pyrosequencing techniques
Creators: name: ARMENIA, DANIELE
Publisher: name: Università degli Studi di Roma "Tor Vergata"
Year: 2010
Subjects: subject: HIV-1 Integrase; HIV drug resistance; mutations; minor variants; antiretroviral therapy
subject: HIV-1 Integrase
subject: HIV drug resistance
subject: mutations
subject: minor variants
subject: antiretroviral therapy

description: 22. ciclo
description: 1 Introduction 1.1 HIV 1.1.1 Morphology 1.1.2 Genome 1.1.3 Replication 1.1.4 Pathogenesis 1.1.5 Epidemiology 1.2 Antiretroviral therapy 1.2.1 Protease inhibitors (PIs) 1.2.2 Reverse transcriptase inhibitors (NRTIs/NNRTIs) 1.2.3 Integrase inhibitors (INIs) 1.2.4 Fusion inhibitors (FIs) 1.2.5 CCR5 inhibitors 1.3 Drug resistance 1.3.1 Mechanism of drug resistance 1.3.1a PIs 1.3.1b NRTIs 1.3.1c NNRTIs 1.3.1d INIs 1.3.1e FIs 1.3.1f CCR5 inhibitors 1.4 Resistance test 1.4.1 Clinical importance of performing resistance test 1.4.2 Phenotypic test 1.4.3 Genotipic test 1.4.4 Interpretation of genotypic resistance profile 1.4.4a PIs 1.4.4b NRTIs 1.4.4c NNRTIs 1.4.4d INIs 73 1.4.5e FIs 75 1.4.5f CCR5 inhibitors 1.5 Rational of work. - 2 Methods 2.1 Clonal analysis 2.1.1 RNA isolation, cDNA synthesis and PCR 2.1.2 Cloning of RT-IN amplicons into HXB2D-based RT-IN deleted backbone 2.1.3 IN site-directed mutant (SDM) plasmids 2.1.4 Production of replication-competent recombinant viruses 2.1.4 Drug susceptibility testing of recombinant viruses (phenotyping) 2.1.5 IN sequencing 2.1.6 Mutations 2.1.7 Replication capacity assays 2.1.8 Statistical analysis 2.2 Population sequencing 2.2.1 Patients 2.2.2 IN Sequencing 2.2.3 Phylogenetic analyses 2.2.4 Mutations 2.2.5 Genotypic sensisitive score 2.2.5 Statistical analysis 2.3 Ultra-Deep-454 Pyrosequencing 2.3.1 Patients 2.3.2 Massively parallel sequencing 2.3.3 Mutations 2.3.4 Poylogenetic analyses 2.3.6 Cloning of IN amplicons into HXB2D-based IN deleted backbone 2.3.7 IN site-directed mutant (SDM) plasmids 2.3.8 Statistical analysis. - 3 Results 3.1 Secondary HIV-1 Integrase Resistance Mutations, Found as Minority Quasispecies in Integrase Therapy Naive Patients, Have Little or no Effect on Susceptibility to Integrase Inhibitors 3.1.1 Production of clonal recombinant virus stocks 3.1.2 Determination of raltegravir and elvitegravir susceptibility of clonal RT-IN recombinant viruses 3.1.3 Genotypic characterization of mutations associated with resistance to raltegravir and Elvitegravir 3.1.4 Phenotypic characterization of mutations associated with resistance to raltegravir and Elvitegravir 3.1.5 Susceptibility to raltegravir and elvitegravir of site direct mutant viruses carrying primary and secondary resistance IN mutations 3.1.6 Genotypic characterization of RT mutations in RT-IN recombinant viruses 3.1.7 Replication capacity of recombinant clonal viruses 3.2 Impact of baseline HIV-1 integrase polymorphisms with virological outcome in patients starting a Raltegravir-containing regimen 3.2.1 Efficacy of raltegravir containing regimen at 24 weeks of treatment 3.2.2 Prevalence of integrase baseline polymorphisms according with subtype 3.2.3 Prevalence of integrase polymorphisms and raltegravir resistance mutations according with subtype and virologic response 3.2.4 Independent predictors of virologic success at 24 weeks of raltegravir treatment by univariate and multivariate logistic regression. 3.2.5. Prevalence of T125A mutations according with raltegravir treatment 3.3. Primary Mutations Associated with Resistance to Raltegravir are not Detectable by Pyrosequencing in Integrase-Inhibitors Naïve Patients 3.3.1 UDPS coverage and sample size results 3.1.2 Prevalence of baseline raltegravir resistance associated mutation detected by Population-sequencing 3.1.3 Comparison of mutation detectability with population sequencing and UDPS 3.1.4 Baseline prevalence of raltegravir resistance mutations detected by UDPS according to virologic response at 24 weeks of raltegravir containing regimen. 3.1.5 Prevalence of raltegravir resistance mutations over time by UDPS 3.1.6 Phylogenetic analyses of haplotypes evolution during raltegravir treatment of 4 representative failing patients 3.1.7 Phenotypic resistance associated with resistance mutations harbored at failure 3.1.8 Phenotypic resistance profile and replication capacity of viruses carrying Y143C/R and/or N155H mutations. - 4 Discussion 5 Summary and conclusions 6 Acknowledgments 7 References