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1
Lin, Kuan-Hung. "Viral Proteases as Drug Targets and the Mechanisms of Drug Resistance: A Dissertation." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/841.
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Viral proteases have been shown to be effective targets of anti-viral therapies for human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, under the pressure of therapy including protease inhibitors, the virus evolves to select drug resistance mutations both in the protease and substrates. In my thesis study, I aimed to understand the mechanisms of how this protease−substrate co-evolution contributes to drug resistance. Currently, there are no approved drugs against dengue virus (DENV); I investigated substrate recognition by DENV protease and designed cyclic peptides as inhibitors targeting the prime site of dengue protease.
First, I used X-ray crystallography and subsequent structural analysis to investigate the molecular basis of HIV-1 protease and p1-p6 substrate coevolution. I found that co-evolved p1-p6 substrates rescue the HIV-1 I50V protease’s binding activity by forming more van der Waals contacts and hydrogen bonds, and that co-evolution restores the dynamics at the active site for all three mutant substrates.
Next, I used aprotinin as a platform to investigate DENV protease–substrate recognizing pattern, which revealed that the prime side residues significantly modulate substrate affinity to protease and the optimal interactions at each residue position. Based on these results, I designed cyclic peptide inhibitors that target the prime site pocket of DENV protease. Through optimizing the length and sequence, the best inhibitor achieved a 2.9 micromolar Ki value against DENV3 protease. Since dengue protease does not share substrate sequence with human serine proteases, these cyclic peptides can be used as scaffolds for inhibitor design with higher specificity.
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Lin, Kuan-Hung. "Viral Proteases as Drug Targets and the Mechanisms of Drug Resistance: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/841.
Full textAbstract:
Viral proteases have been shown to be effective targets of anti-viral therapies for human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, under the pressure of therapy including protease inhibitors, the virus evolves to select drug resistance mutations both in the protease and substrates. In my thesis study, I aimed to understand the mechanisms of how this protease−substrate co-evolution contributes to drug resistance. Currently, there are no approved drugs against dengue virus (DENV); I investigated substrate recognition by DENV protease and designed cyclic peptides as inhibitors targeting the prime site of dengue protease.
First, I used X-ray crystallography and subsequent structural analysis to investigate the molecular basis of HIV-1 protease and p1-p6 substrate coevolution. I found that co-evolved p1-p6 substrates rescue the HIV-1 I50V protease’s binding activity by forming more van der Waals contacts and hydrogen bonds, and that co-evolution restores the dynamics at the active site for all three mutant substrates.
Next, I used aprotinin as a platform to investigate DENV protease–substrate recognizing pattern, which revealed that the prime side residues significantly modulate substrate affinity to protease and the optimal interactions at each residue position. Based on these results, I designed cyclic peptide inhibitors that target the prime site pocket of DENV protease. Through optimizing the length and sequence, the best inhibitor achieved a 2.9 micromolar Ki value against DENV3 protease. Since dengue protease does not share substrate sequence with human serine proteases, these cyclic peptides can be used as scaffolds for inhibitor design with higher specificity.
3
Wilbe, Karin. "Genetic dynamics of HIV-1: recombination, drug resistance and intrahost evolution /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-959-5/.
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Ozen, Aysegul. "Structure and Dynamics of Viral Substrate Recognition and Drug Resistance: A Dissertation." eScholarship@UMMS, 2005. http://escholarship.umassmed.edu/gsbs_diss/677.
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Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors.
5
Ozen, Aysegul. "Structure and Dynamics of Viral Substrate Recognition and Drug Resistance: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/677.
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Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors.
6
Lockbaum, Gordon J. "Molecular Mechanisms of Resistance and Structure-Based Drug Design in Homodimeric Viral Proteases." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1072.
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Drug resistance is a global health threat costing society billions of dollars and impacting millions of lives each year. Current drug design strategies are inadequate because they focus on disrupting target activity and not restricting the evolutionary pathways to resistance. Improved strategies would exploit the structural and dynamic changes in the enzyme–inhibitor system integrating data from many inhibitors and variants.
Using HIV-1 protease as a model system, I aimed to elucidate the underlying resistance mechanisms, characterize conserved protease-inhibitor interactions, and generate more robust inhibitors by applying these insights. For primary mechanisms of resistance, comparing interactions at the protease–inhibitor interface showed how specific modifications affected potency. For mutations distal to the active site, molecular dynamics simulations were necessary to elucidate how changes propagated to reduce inhibitor binding. These insights informed inhibitor design to improve potency against highly resistant variants by optimizing hydrogen bonding. A series of hybrid inhibitors was also designed that showed excellent potency by combining key moieties of multiple FDA-approved inhibitors. I characterized the structural basis for alterations in binding affinity in HIV-1 protease both from mutations and inhibitors.
I applied these strategies to HTLV-1 protease, a potential drug target. I identified the HIV-1 inhibitor darunavir as a viable scaffold and evaluated analogues, leading to a low-nanomolar compound with potential for optimization. Hopefully, insights from this thesis will lead to the development of potent HTLV-1 protease inhibitors. More broadly, these inhibitor design strategies are applicable to other rapidly evolving targets, thereby reducing drug resistance rates in the future.
7
Svedhem, Johansson Veronica. "Kinetics of HIV-1 drug resistance mutations in vivo /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-671-9/.
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Pugach, Pavel. "The evolutionary response of the HIV-1 ENV complex to selection pressures in vitro /." Access full-text from WCMC:, 2007. http://proquest.umi.com/pqdweb?did=1428842531&sid=4&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Zephyr, Jacqueto. "Robust Drug Design Strategies and Discovery Targeting Viral Proteases." eScholarship@UMMS, 2021. https://escholarship.umassmed.edu/gsbs_diss/1157.
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Viral proteases play crucial roles in the life cycle and maturation of many viruses by processing the viral polyprotein after translation and in some cases cleaving host proteins associated with the immune response. The essential role of viral proteases makes them attractive therapeutic targets. In this thesis, I provide an introductory summary of viral proteases, their structure, mechanism, and inhibition, while the breadth of this thesis focuses on the Hepatitis C virus (HCV) NS3/4A and Zika virus (ZIKV) NS2B/NS3 viral proteases.
HCV NS3/4A protease inhibitors (PIs) have become a mainstay in combination therapies. However, drug resistance remains a major problem against these PIs. In this thesis, I applied insights from the HCV substrate envelope (SE) model to develop strategies for designing PIs that are less susceptible to resistance. Also, I used the HCV NS3/4A protease as a model system to decipher the molecular mechanism and role of fluorination in HCV PIs potency and drug resistance. The drug design strategies described in this thesis have broad applications in drug design.
The ZIKV is an emerging global threat, and currently, with no treatment available. In this thesis, I described the discovery, biochemical and antiviral evaluation of novel noncompetitive quinoxaline-based inhibitors of the ZIKV NS2B/NS3 protease. The inhibitors are proposed to interfere with NS2 binding to NS3, thereby preventing the protease from adopting the closed and active conformation. The inhibitors from this work will serve as lead compounds for further inhibitor development toward the goal of developing antivirals.
10
Malik, S. "The role of the HIV-1 reverse transcriptase mutation H208Y to drug resistance and viral fitness." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1207313/.
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Accessory mutations are thought to arise alongside major or primary drug resistance mutations in order to augment resistance, restore viral fitness, or both. The H208Y mutation in the HIV-1 reverse transcriptase (RT) gene was hypothesised to be an accessory mutation. This thesis characterises the H208Y mutation in terms of linkage of H208Y to other major and accessory resistance mutations, and examines two phenotypic aspects, drug susceptibility and viral fitness. The HIV Resistance Database held at the Royal Free Hospital was searched for genotypes containing the H208Y mutation. The prevalence of H208Y in antiretroviral treatment naïve and treatment experienced patients was 5/3783 (0.1%) and 12/1304 (0.9%), respectively, indicating a high degree of conservation of position 208 in wild type virus and an increase in prevalence under selective drug pressure. Four patients were chosen to conduct further analysis of virus with H208Y, comprising a treatment naïve patient with subtype B virus, and three treatment experienced patients harbouring subtype A, subtype B and subtype C virus respectively. The RT gene from these patients was cloned and sequenced. H208Y was found to be associated with the thymidine analogue mutations (TAMs), particularly mutations at positions D67, T215 and K219. H208Y was always associated with accessory mutations at positions V35, K122 and T200. Recombinant viruses containing patient derived RT genes with and without H208Y were constructed to examine the impact of H208Y on drug susceptibility and viral fitness. A multiple cycle drug susceptibility assay showed that H208Y conferred a reduced susceptibility to the nucleotide RT inhibitor tenofovir in the context of subtype B wild type RT and subtype B RT containing TAMs. Growth competition assays were used to examine the fitness effects of H208Y using allele-specific PCR to differentiate between competing strains with and without H208Y. In the context of subtype B wild type RT, H208Y conferred a reduced viral fitness both in the presence and absence of drug. The effect may be proposed to contribute to the overall high degree of conservation of position 208. In contrast, H208Y did not appear to impact on viral fitness in the context of subtype B and subtype A RT containing TAMs.
11
Freeman, Mark. "Synchronization of Viral Lifecycle Length to Antiviral Drug Dosage Schedules and the Emergence of "Cryptic Resistance''." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17417584.
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Viral infections, such as HIV, are often treated with orally administered antiviral medications that are dosed at particular intervals, leading to periodic drug levels and hence periodic inhibition of viral replication. These drugs generally bind to viral proteins and inhibit particular steps in the viral lifecycle, and resistance often evolves due to point mutations in the virus that prevent the drug from binding its target. However, it has been proposed (Wahl \& Nowak, Proc Roy Soc B, 2000) that a completely different ``cryptic'' mechanism for resistance could exist: the virus population may evolve towards synchronizing its lifecycle with the pattern of drug treatment. If the lifecycle of the virus is a multiple of the dosing interval, it is possible that over time the bulk of the virus population will replicate during trough concentrations of the drug. In this thesis, we use stochastic mathematical models of viral dynamics to demonstrate that cryptic resistance could plausibly provide a powerful fitness advantage to a wide variety of viral strains whose expected lifecycle times are slightly less than the expected time between doses of an antiviral drug, allowing them to survive drug regimes that would otherwise drive infected cell populations to extinction. This in turn suggests that continuously-administered antiviral drug treatments may be significantly more effective than periodically-administered treatments in combatting viral infections.Applied Mathematics
12
Okleberry, Kevin M. "Metabolism of Selected Antiviral Agents in Cells Infected with Drug-Resistant and Wild-Type Strains of Murine Cytomegalovirus." DigitalCommons@USU, 1995. https://digitalcommons.usu.edu/etd/4657.
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Resistance of human viral pathogens to various antiviral drugs is a serious medical problem. Two modes of drug resistance in cytomegalovirus infections have been observed, the first being altered (decreased) drug metabolism by the infected cells, and the second reduced sensitivity of the viral deoxyribonucleic acid polymerase enzyme to the active form of the drug. Mice infected with the murine cytomegalovirus have been used extensively as an animal model for the human cytomegalovirus, and drug-resistant strains in this model have been identified. To better understand the mode of drug resistance of the virus, the metabolism of two antiviral drugs, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (ganciclovir) and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (cidofovir), was studied in cells infected with the virus.
The degree of resistance of the mutant virus strain to these two drugs and also to the drug phosphonoformic acid (foscarnet) was measured in viral plaque reduction assays. The resistant strain was 14-,4-, and 11-fold less sensitive to the drugs ganciclovir, foscarnet, and cidofovir, respectively, than a sensitive (wildtype) strain
Metabolism of the antiviral drugs ganciclovir and cidofovir was studied in C127I mouse mammary tumor cells infected with the mutant strain. Uninfected C127I cells and C127I cells infected with the sensitive strain of murine cytomegalovirus were used as controls. The cells were treated with tritium-labeled ganciclovir or cidofovir and studied under a variety of parameters. Among these were duration of treatment, multiplicity of infection, and concentration of compound. After incubating, the cells were acid extracted and analyzed with high-pressure liquid chromatography. The radioactivity of each sample was measured on a scintillation counter and converted into picomoles of drug per million cells.
No significant difference was observed between the virus strains in terms of metabolism or catabolism of the two drugs. This effect remained constant, even when controlling for parameters such as the amount of virus infecting each cell, duration of treatment, or concentration of drug. Based on these results, it appears that the mode of resistance in this mutant strain of virus to ganciclovir and cidofovir is not due to an alteration in metabolism of these two compounds by infected cells. Thus, it is proposed that drug resistance in this mutant strain of virus is due to altered viral deoxyribonucleic acid polymerase function.
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Eriksen, Jaran. "Managing childhood malaria in rural Tanzania : focusing on drug use and resistance /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-678-6/.
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Soumana, Djade I. "Hepatitis C Virus: Structural Insights into Protease Inhibitor Efficacy and Drug Resistance: A Dissertation." eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/803.
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The Hepatitis C Virus (HCV) is a global health problem as it afflicts an estimated 170 million people worldwide and is the major cause of viral hepatitis, cirrhosis and liver cancer. HCV is a rapidly evolving virus, with 6 major genotypes and multiple subtypes. Over the past 20 years, HCV therapeutic efforts have focused on identifying the best-in-class direct acting antiviral (DAA) targeting crucial components of the viral lifecycle, The NS3/4A protease is responsible for processing the viral polyprotein, a crucial step in viral maturation, and for cleaving host factors involved in activating immunity. Thus targeting the NS3/4A constitutes a dual strategy of restoring the immune response and halting viral maturation. This high priority target has 4 FDA approved inhibitors as well as several others in clinical development. Unfortunately, the heterogeneity of the virus causes seriously therapeutic challenges, particularly the NS3/4A protease inhibitors (PIs), which suffer from both the rapid emergence of drug resistant mutants as well as a lack of pan-genotypic activity.
My thesis research focused on filling two critical gaps in our structural understanding of inhibitor binding modes. The first gap in knowledge is the molecular basis by which macrocyclization of PIs improves antiviral activity. Macrocycles are hydrophobic chains used to link neighboring chemical moieties within an inhibitor and create a structurally pre-organized ligand. In HCV PIs, macrocycle come in two forms: a P1 - P3 and P2 - P4 strategy. I investigated the structural and thermodynamic basis of the role of macrocyclization in reducing resistance susceptibility. For a rigorous comparison, we designed and synthesized both a P1 - P3 and a linear analog of grazoprevir, a P2 - P4 inhibitor. I found that, while the P2 - P4 strategy is more favorable for achieving potency, it does not allow the inhibitor sufficient flexibility to accommodate resistance mutations. On the other hand, the P1 - P3 strategy strikes a better balance between potency and resistance barrier.
The second gap my thesis addresses is elucidating the structural basis by which highly potent protease inhibitors function in genotype 1 but not in genotype 3, despite having an 87% sequence similarity. After mapping the amino acids responsible for this differential efficacy in genotypes 1 and 3, I engineered a 1a3a chimeric protease for crystallographic studies. My structural characterization of three PIs in complex with both the 1a3a and genotype 1 protease revealed that the loss of inhibitor efficacy in the 1a3a and GT-3 proteases is a consequence of disrupted electrostatic interactions between amino acids 168 and 155, which is critical for potent binding of quinoline and isoindoline based PIs. Here, I have revealed details of molecular and structural basis for the lack of PI efficacy against GT-3, which are needed for design of pan-genotypic inhibitors.
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Matthew, Ashley N. "Targeting Drug Resistance In HCV NS3/4A Protease: Mechanisms And Inhibitor Design Strategies." eScholarship@UMMS, 2018. https://escholarship.umassmed.edu/gsbs_diss/969.
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The Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs) have become a mainstay of newer all-oral combination therapies. Despite improvements in potency of this inhibitor class, drug resistance remains a problem with the rapid emergence of resistance-associated substitutions (RASs). In this thesis I elucidate the molecular mechanisms of drug resistance for PIs against a resistant variant and apply insights toward the design of inhibitors with improved resistance profiles using structural, biochemical and computational techniques. Newer generation PIs retain high potency against most single substitutions in the protease active site by stacking on the catalytic triad. I investigated the molecular mechanisms of resistance against the Y56H/D168A variant. My analysis revealed that the Y56H substitution disrupts these inhibitors’ favorable stacking interactions with the catalytic residue His57.
To further address the impact of drug resistance, I designed new inhibitors that minimize contact with known drug resistance residues that are unessential in substrate recognition. The initially designed inhibitors exhibited flatter resistance profiles than the newer generation PIs but lost potency against the D168A variant. Finally, I designed inhibitors to extend into the substrate envelope (SE) and successfully regained potency against RAS variants maintaining a flat profile. These inhibitors both pack well in the enzyme and fit within the SE. Together these studies elucidate the molecular mechanisms of PI resistance and highlight the importance of substrate recognition in inhibitor design. The insights from this thesis provide strategies toward the development of diverse NS3/4A PIs that may one day lead to the eradication of HCV.
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To, Wai-chi Sabrina, and 杜維之. "Molecular characterization of HIV-1 in Hong Kong : a study of drug resistance mutations, tropism and viral evolution." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/207184.
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Human immunodeficiency virus type 1 (HIV-1) infection is a life-long threat which cannot be prevented by vaccination or completely cured by antiretroviral (ARV) treatment. The establishment of genotypic resistant testing (GRT) greatly improved the infection management and provided further guidelines on ARV strategies. The current study aimed to utilize the accumulated GRT data to investigate the HIV-1 molecular epidemiology, trends of drug resistance mutations (DRMs) among local patients and intra-host viral evolution. In order to maximize the therapeutic options for HIV-1 patients, the genotypic study also extended to two relatively new ARV classes for determination of integrase polymorphisms and tropism prevalence.
From 1994 through 2013, a total of 3,108 plasma samples were available from 2,475 HIV-1 positive patients for epidemiological and viral investigations. The predominant genotypes in Hong Kong were subtype B (42.1%) and CRF01_AE (39.7%). Other genotypes including subtypes A1, C, D, F1, G, CRF02_AG, CRF06_cpx, CRF07_BC, CRF08_BC and CRF12_BF were also identified. Though the phylogenetic studies of subtype B and CRF01_AE identified several sporadic distinct clusters, their transmissions had been widely established between all local risk groups. In contrast, most of the non-B and non-AE cases were infected outside Hong Kong or circulated within non-Chinese population. However, one of the clusters in CRF07_BC suggested that this genotype might have been established quite well among local Chinese men-who-have-sex-with-men community. Routine GRT also provided valuable longitudinal data to study intra-host viral evolution. The observed intra-host evolutionary rates in CRF01_AE variants (11.68 x 〖10〗^(-4), IQR: 8.87 – 20.54 x 〖10〗^(-4) substitutions per site per year) was significantly higher than subtype B variants (5.27 x 〖10〗^(-4), IQR: 3.32 – 8.01 x 〖10〗^(-4) substitutions per site per year).
Of 2,157 treatment-naïve patients included in this study, 4.4% of them were found carrying surveillance protease (PR) or reverse transcriptase (RT) DRMs. More importantly, half of the GRT data from 407 treatment-failure patients were potentially resistant to PR or RT inhibitors. The introduction of integrase inhibitors and CCR5 antagonists provided new insights and era for treating HIV-1 patients who were resistant to PR or RT inhibitors. The integrase GRT results showed that there were no major DRMs observed in integrase inhibitors-naïve patients. The distribution of integrase polymorphic sites between subtype B and CRF01_AE was significantly differed. On the other hand, the prevalence of X4/Dual-Mixed tropism in CRF01_AE variants was always significantly higher than subtype B, regardless of the interpretation algorithms and treatment history.
In conclusion, this study demonstrated the HIV-1 molecular epidemiology and intra-host viral evolution of Hong Kong in the last two decades by utilizing the GRT data obtained from the ARV surveillance program. The in-house integrase GRT and genotypic tropism test were as well evaluated. The high prevalence of X4/Dual-Mixed tropism in CRF01_AE isolates would definitely limit the salvage therapeutic options for CRF01_AE-infected patients. Summarizing these findings, it is possible to project that CRF01_AE virus has unique characteristics from subtype B and should be investigated further in terms of disease progression and replicative capacity.published_or_final_version
Microbiology
Doctoral
Doctor of Philosophy
17
Dolling, D. I. "HIV-1 viral load outcomes and the evolution of drug-resistance in low-income settings without virological monitoring." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1557352/.
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WHO guidelines recommend viral load monitoring for all HIV-1 positive patients on antiretroviral therapy (ART). However, few low-income countries have virological monitoring widely available, and patients may remain on virologically failing regimens. This could compromise future ART through the accumulation of drug resistance mutations and result in worse long-term clinical outcomes. The DART trial was conducted in Uganda and Zimbabwe and compared clinically driven monitoring with or without routine CD4 measurement in ART-naïve adult patients. Annual plasma viral load was retrospectively measured for 1,762 patients. This thesis investigates how no laboratory monitoring impacts virological failure and the development of drug resistance. Time to persistent virological failure was analysed, and analytical weights were calculated to correct for non-random sampling. The long-term durability of first-line ART was remarkable; 21% of patients on an NRTI-NNRTI regimen and 40% on a triple-NRTI regimen experienced persistent virological failure by 240 weeks. Routine CD4 monitoring did not reduce virological failure. Deaths after 48 weeks of ART are widely assumed to be due to virological failure or non-adherence. Analyses revealed that a surprisingly high number of these deaths (40%) occurred without virological criteria for treatment switch being met. Routine CD4 monitoring reduced the rate of death with virological failure but did not impact deaths with virological suppression. Cross-sectional analyses quantified HIV-1 drug resistance at the end of first-line ART. On NRTI-NNRTI regimens, 88% had NRTI resistance, and 66% had NNRTI resistance. Routine CD4 monitoring did not reduce the prevalence or extent of drug resistance. The order and rate of HIV-1 drug resistance mutations were explored using repeated genotypes within patients. On NRTI-NNRTI regimens, NRTI and NNRTI mutations developed at a rate of 0.96 and 0.21 per year respectively. Mutagenic tree models demonstrated that ART regimen influenced the order and rate in which mutations occurred.
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Lindström, Anna. "Resistance to antiviral drugs in HIV and HBV /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-239-X/.
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Romano, Keith P. "Mechanisms of Substrate Recognition by HCV NS3/4A Protease Provide Insights Into Drug Resistance: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/554.
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HCV afflicts many millions of people globally, and antiviral therapies are often ineffective and intolerable. The Food and Drug Administration approved the HCV protease inhibitors telaprevir and boceprevir in May 2011, marking an important milestone in anti-HCV research over the past two decades. Nevertheless, severe drug side effects of combination therapy – flu-like symptoms, depression and anemia – limit patient adherence to treatment regimens. The acquisition of resistance challenges the long-term efficacy of antiviral therapies, including protease inhibitors, as suboptimal dosing allows for the selection of drug resistant viral variants. A better understanding of the molecular basis of drug resistance is therefore central to developing future generation protease inhibitors that retain potency against a broader spectrum of HCV strains.
To this end, my research characterizes the molecular basis of drug resistance against HCV protease inhibitors. Chapter II defines the mode of substrate recognition by the common volume shared by NS3/4A substrate products – the substrate envelope. Chapter III then correlates patterns of drug resistance to regions where drugs protrude from the substrate envelope. Lastly, Chapter IV elucidates the molecular underpinnings of resistance against four leading protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – and provides practical approaches to designing novel drugs that are less susceptible to resistance. I ultimately hope my work appeals to the broader biomedical community of virologists, medicinal chemists and clinicians, who struggle to understand HCV and other human pathogens in the face of rapid disease evolution.
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LIMA, Kledoaldo Oliveira De. "Características filogenéticas, epidemiológicas, laboratoriais e evolutivas dos subtipos B e não-B do HIV-1 em Pernambuco – Nordeste do Brasil." Universidade Federal de Pernambuco, 2015. https://repositorio.ufpe.br/handle/123456789/15666.
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CAPES
Uma grande heterogeneidade da epidemia pelo HIV-1 é observada no Brasil, onde prevalecem os subtipos B, F1 e C e os recombinantes BF e BC. O objetivo principal deste estudo foi caracterizar as cepas do HIV-1 circulantes no estado de Pernambuco, Nordeste do Brasil, através de análises filogenéticas, avaliando-se as características sociodemográficas, laboratoriais e evolutivas entre os subtipos B e não-B. As sequências da região pol do HIV-1, que totalizaram 169 amostras, foram obtidas de dois estudos anteriores. Todos os pacientes eram maiores de 18 anos e virgens de terapia antirretroviral. O Alinhamento e a edição manual das sequências foram realizados pelo CLUSTAL X e BioEdit software, respectivamente. Para as inferências filogenéticas e de recombinação gênica foram utilizados os softwares MEGA 5 e SIMPLOT, respectivamente. Pesquisas sorológicas para determinação das co-infecções foram realizadas para os seguintes agentes infecciosos: HBV, HCV, HTLV e sífilis, pelo método de quimiluminescência. Na região analisada (pol), os resultados mostraram uma grande frequência do subtipo F do HIV-1 (31.4%) e a circulação de uma cepa H e AG. As frequências dos subtipos B e C foram 60.9% e 1.2%, respectivamente. Foram identificados um recombinante BC e 8 recombinantes BF (4.7%), com estruturas genômicas diferenciadas. Co-infecção HIV-HBV foi mais frequente entre homens que fazem sexo com homens (HSH) portadores do subtipo B do HIV-1, enquanto que co-infecção HIV-sífilis foi associado a HSH com subtipos não-B. O subtipo B foi associado ao sexo masculino, a uma maior carga vira, maior escolaridade e menor contagem de células T CD4+. Houve uma baixa frequência de mutações de resistência transmitidas (2.96%). Códons sob pressão seletiva positiva são mais frequentes em contagem de células T CD4+ ≥ 200 e em mulheres heterossexuais. Nossos resultados demonstram que a epidemia do HIV-1 em Pernambuco se caracteriza por uma alta proporção de subtipos não-B circulantes, o que revela a importância no monitoramento e melhor conhecimento do papel destas variantes na epidemia, no tratamento antirretroviral, formulação de vacinas, progressão à doença e transmissibilidade.
A great heterogeneity of HIV-1 epidemic is observed in Brazil, where subtypes B, F1 and C and recombinant forms BF prevails and BC. The aim of study was to characterize HIV-1 strains circulating in the state of Pernambuco, northeastern Brazil, through phylogenetic analysis, evaluating also the socio-demographic, laboratory and evolutionary characteristics between subtypes B and non-B. The sequences with pol region of HIV-1, totaling 169 samples were obtained from two previous studies. All patients were over 18 year old and antiretroviral naïve therapy. The alignment and manual editing of the sequences were performed by CLUSTAL X and BioEdit software, respectively. For the phylogenetic and genetic recombination inferences were used MEGA 5 and SIMPLOT software, respectively. Serological assaus of co-infections were performed for the following infectious agents: HBV, HCV, HTLV, and syphilis by chemiluminescence. In the analyzed region (pol), the results showed a high frequency of the HIV-1 subtype F (31.4%) and a strain H and AG. The frequency of the subtypes B and C were 60.9% and 1.2%, respectively. They identified a recombinant BC and eight BF recombinant (4.7%) with different genomic structures. Co-infection HIV-HBV was more frequent among men who have sex with men (MSM) with HIV-1 subtype B, while co-infection HIV-syphilis was associated with MSM with subtypes non-B. The subtype B was associated with male gender, higher viral load, higher education and lower T cells count. There was a low frequency of transmitted resistance mutations (2.96%). Codons under positive selection pressure are more common in T cells count ≥ 200 and heterosexual women. Our results demonstrate that the HIV-1 epidemic in Pernambuco is characterized by a high proportion of circulating non-B subtypes, which shows the importance of monitoring and better understanding of the role of these variants in epidemic in antiretroviral therapy, vaccine formulation, disease progression and transmissibility.
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Nguyen, Albert Thu. "The molecular mechanism of action of bevirimat : a prototype HIV-1 maturation inhibitor /." Oklahoma City : [s.n.], 2009.
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Prachanronarong, Kristina L. "Understanding Drug Resistance and Antibody Neutralization Escape in Antivirals: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/840.
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Antiviral drug resistance is a major problem in the treatment of viral infections, including influenza and hepatitis C virus (HCV). Influenza neuraminidase (NA) is a viral sialidase on the surface of the influenza virion and a primary antiviral target in influenza. Two subtypes of NA predominate in humans, N1 and N2, but different patterns of drug resistance have emerged in each subtype. To provide a framework for understanding the structural basis of subtype specific drug resistance mutations in NA, we used molecular dynamics simulations to define dynamic substrate envelopes for NA to determine how different patterns of drug resistance have emerged in N1 and N2 NA. Furthermore, we used the substrate envelope to analyze HCV NS3/4A protease inhibitors in clinical development. In addition, influenza hemagglutinin (HA) is a primary target of neutralizing antibodies against influenza. Novel broadly neutralizing antibodies (BnAbs) against the stem region of HA have been described and inhibit several influenza viral subtypes, but antibody neutralization escape mutations have emerged. We identified potential escape mutations in broadly neutralizing antibody F10 that may impact protein dynamics in HA that are critical for function. We also solved crystal structures of antibody fragments that are important for understanding the structural basis of antibody binding for influenza BnAbs. These studies can inform the design of improved therapeutic strategies against viruses by incorporating an understanding of structural elements that are critical for function, such as substrate processing and protein dynamics, into the development of novel therapeutics that are robust against resistance.
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Angelis, Daniela Souza Araujo de. "Avaliação do perfil de resistência genotípica aos anti-retrovirais de crianças infectadas pelo HIV-1 mantendo supressão viral prolongada em vigência de tratamento." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/5/5134/tde-21062007-154402/.
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O tratamento de indivíduos infectados pelo HIV-1 com terapia anti-retroviral (ARV) pode reduzir a viremia plasmática abaixo dos limites de detecção dos ensaios atuais em muitos pacientes, porém difícil de ser alcançada em crianças na vida real. Falha em alcançar ou manter a supressão da replicação viral está geralmente associada com o desenvolvimento de vírus resistentes a drogas. Nós investigamos o perfil de resistência genotípica em crianças com supressão viral prolongada (< 400 cópias/mL de RNA viral plasmático) em vigência de tratamento anti-retroviral. Nós obtivemos 32 amostras de células mononucleares do sangue periférico (do inglês PBMC) de 16 crianças do CEADIPe - UNIFESP quem tinham tido carga viral indetectável por 12 meses ou mais, em dois momentos: a primeira amostra na inclusão e a segunda após mínimo de 9 meses de acompanhamento. A análise das seqüências foi realizada em vírus isolado de PBMC pelo \"ABI PRISM 377 sequencer\" (Applied Biosystems, USA). Dentre as principais características da população do estudo encontramos: mediana da idade na inclusão de 11 (6-15 anos); esquemas terapêuticos com 2 inibidores da transcriptase reversa análogos nucleosídeo (ITRN) + 1 inibidor da protease (IP) ou 2 ITRN + 1 inibidor da transcriptase reversa não-nucleosídeo (ITRNN) ou 2 ITRN + 2 IP + 1 ITRNN ou 2 ITRN + 2 IP ou 2 ITRN; mediana de células CD4 (cél/mm 3 ) de 1016 (347- 2588) e 938 (440-3038) no primeiro e segundo momentos, respectivamente; classificação clínica (CDC 1994): N = 1, A = 3, B = 6; e classificação imune (CI): CI 1 = 4, CI 2 = 6, CI 3 = 6. O tempo médio de seguimento foi 15 (9 - 27) meses a partir da inclusão. Seis (37,5%) e 7 (43,75%) dos 16 pacientes mostraram no mínimo uma mutação associada aos ITRN, na primeira e na segunda amostra, respectivamente. Dois dos dezesseis (12,5%) apresentaram mutações associadas aos ITRNN na primeira amostra e 3/16 (18,75%) na segunda. Além disso, 14/16 (87,5%) mostraram pelo menos uma mutação associada aos IP nos dois momentos. A despeito do tratamento com drogas anti-retrovirais potentes e supressão do RNA do HIV-1 no plasma a níveis indetectáveis por vários meses, resistência parcial à terapia pode ter resultado primariamente de arquivos de vírus ou refletir precocemente condições sub-ótimas de tratamento.Treatment of HIV1-infected individuals with antiretroviral therapy (ARV) can reduce plasma viremia to below the limits of detection of current assays in many patients, although it is difficult to happen to children in real life. Failure to achieve or maintain suppression of viral replication is often associated with the development of drug-resistant virus. We investigated genetic resistance profiles of low-level plasma HIV-1 in children with prolonged viral suppression (<400copies/mL of plasma HIV-1 RNA) while receiving ARV. We obtained 32 samples of peripheral-blood mononuclear cells (PBMC) from 16 children from CEADIPe - UNIFESP who had had undetectable viral load for 12 months or more, at two moments: first sample at the inclusion and second after a minimum 9-months follow-up time. Sequence analysis was performed on virus isolated from PBMC by \"ABI PRISM 377 sequencer\" (Applied Biosystems, USA). The main characteristics of the study population were: median age baseline = 11 (6-15 years); drug combinations = 2 nucleoside reverse transcriptase inhibitor (NRTI) + 1 protease inhibitor (PI) or 2 NRTI + 1 non-nucleoside reverse transcriptase (NNRTI) or 2 NRTI + 2 PI + 1 NNRTI or 2 NRTI + 2 PI or 2 NRTI; median CD4 cell count (cells/mm 3 ) = 1016 (347-2588) and 938 (440-3038) at first and second time points, respectively; clinic classification (CDC 1994): N = 1, A = 3, B = 6; and immune classification (IC): IC 1 = 4, IC 2 = 6, IC 3 = 6. The median follow-up time was 15 (9 - 27) months starting from the inclusion. Six (37,5%) and 7 (43,75%) of the 16 patients showed at least one NRTI-associated mutation, in the first and second samples, respectively. Two out of sixteen (12,5%) presented NNRTI-associated mutation at the first moment and 3/16 (18,75%) at the second. In addition, 14/16 (87,5%) showed at least one PI-associated mutation at both moments. Despite treatment with potent antiretroviral drugs and plasma HIV-1 RNA suppression to undetectable levels for several months, partial resistance to therapy may result primarily from archival or contemplate earlier sub optimal treatment conditions.
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Van, Zyl Gert Uves. "The investigation of genotypic antiretroviral drug resistance in the context of the South African national antiretroviral roll-out programme." Thesis, Stellenbosch : Stellenbosch University, 2012. http://hdl.handle.net/10019.1/20254.
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Thesis (PhD)--Stellenbosch University, 2012.ENGLISH ABSTRACT: Introduction: Since the South African public sector antiretroviral roll-out programme started in 2004, the success of antiretroviral combination therapy (cART) has been experienced in terms of survival, prevention of mother-to-child transmission (PMTCT) and quality of life. However, as the programme matures, viral resistance to the constituent drugs will increase. Monitoring antiretroviral drug resistance (ARVDR) should therefore be a priority in the public health approach to HIV treatment. Methods: A cross-sectional investigation of genotypic antiretroviral drug resistance in: a) HIV-infected mothers who were exposed to a PMTCT regimen of short course azidothymidine (AZT) with single dose nevirapine (NVP) during labour. b) HIV-infected adults and children who were cART-naïve (transmitted or initial resistance). c) HIV-infected adults and children who were failing cART (drug-induced or acquired resistance). In case of adults, this includes patients on a first-line, non-nucleoside reverse transcriptase (NNRTI)-based regimen, or on a second-line, protease inhibitor (PI)-based regimen, and in case of children, this includes patients on a first-line PI-based regimen. Results: In mothers who received a PMTCT-regimen that combined AZT and NVP the prevalence of NNRTI resistance mutations was 17.1% (95% CI: 8.7-25.6%). The prevalence of transmitted ARVDR in adults was low, as was initial ARVDR in young children (mostly PMTCT-exposed), except for NNRTI resistance in children who had received NVP as part of PMTCT. Drug-induced resistance was found in adults failing first-line NNRTI-based cART, with 83% having resistance to ≥1 drug. In contrast, adult patients failing second-line PI-based cART had a low prevalence of PI resistance; the predominant reason for failure was poor drug exposure, as detected by measuring lopinavir concentrations in blood plasma and hair samples. In contrast, PI resistance in children was not rare, largely due to historic exposure to un-boosted PIs. This resulted in extensive resistance to PIs and reverse transcriptase inhibitors (RTI) in some children. Conclusions: A combined regimen of short course AZT with intrapartum NVP for PMTCT may, in addition to reducing the risk of neonatal infection, also reduce the risk of NVP resistance in the mothers compared to a regimen of NVP only. In South Africa, the prevalence of transmitted ARVDR remains low relative to industrialised countries, probably as comparatively little time has elapsed since the scale-up of cART. Adults failing first-line cART are likely to respond to second-line cART, without failure due to resistance. However some children with PI and RTI resistance cannot be adequately treated with drugs currently available through the roll-out programme. This emphasizes the urgent need for a rational and science-based approach to managing cART-experienced children, including access to additional drugs to form a third-line paediatric cART regimen.
AFRIKAANSE OPSOMMING: Inleiding: Sedert die begin van die Suid Afrikaanse publieke sektor antiretrovirale uitrol program in 2004 is die sukses van antiretrovirale kombinasie-behandeling (k-ARB) ervaar in terme van oorlewing, voorkoming van moeder na kind oordrag (VMKO) en lewenskwaliteit. Nietemin, sal weerstandigheid teen die middels wat in die antiretrovirale program gebruik word toeneem soos wat die program gevestig raak. Die monitoring van antiretrovirale middel-weerstandigheid is derhalwe ‘n prioriteit in gemeenskap-gesondheid benadering tot MIV behandeling. Metodes: ‘n Deursnit ondersoek van genotipiese antiretrovirale middel-weerstandigheid in: a) MIV-geïnfekteerde moeders wat blootgestel is aan VMKO regimen bestaande uit ‘n kort kursus AZT met ‘n enkeldosis nevirapien (NVP) tydens kraam. b) MIV-geïnfekteerde volwassenes en kinders wat komibinasieterapie-naïef (oorgedraagde of inisiële weerstandigheid) is. c) MIV-geïnfekteerde volwassenes en kinders wat k-ARB faal (middel-geïnduseerde weerstandigheid). In geval van volwassenes, sluit dit pasiënte op ‘n eerste-linie, non-nucleosied tru-transkriptase inhibitor (NNRTI)-regimen, en tweede-linie protease inhibitor (PI)-gebaseerde regimen, en in geval van kinders, sluit dit pasiënte in op ‘n eerste-linie PI-gebaseerde regimen. Resultate: In moeders wat ‘n gekombineerde AZT en NVP VMKO-regimen ontvang het, was die voorkoms van NNRTI weerstandigheid 17.1% (95%-vertrouensinterval: 8.7-25.6%). Die voorkoms van oorgedraagde ARVMW in MIV-geïnfekteerde volwassenes en kinders wat kombinasieterapie-naïef is, was laag, so ook ARVMW in jong kinders (meestal VMKO-blootgestel), behalwe vir non-nukleosied tru-transkriptase inhibitor (NNRT) weerstandigheid in kinders wat NVP ontvang het deur VMKO. Middel-geïnduseerde weerstandigheid was gevind in volwassenes wat die eerste-linie NNRTI-gebaseerde k-ARB gefaal het, met 83% wat weerstandigheid teen ≥1 middel het. Volwassenes wat ‘n tweede-linie protease inhibitor (PI) –gebaseerde k-ARB gefaal het , het ‘n lae voorkoms van PI weerstandigheid, met die oorwegenede oorsaak, swak middel-bloostelling, soos bepaal deur van lopinavir-konsentrasies in bloed plasma en hare. In teenstelling hiermee was PI weerstandigheid nie skaars in kinders nie, hoofsaaklik weens historiese blootstelling an ongeskraagde PI-behandeling. Dit het tot uitgebreide weerstandigheid tot PIs en tru-transkritptase inhibitors (RTI) in sommige kinders gelei. Gevolgtrekkings: ‘n Gekombineerde regimen van ‘n kort kursus AZT met NVP tydens kraam vir VKMO, mag bykomend tot die vermindering die risiko van pasgebore infeksie, ook die kans vir weerstandigheid teen NVP in die moeders verlaag in vergelyking met ‘n regimen van NVP-alleen. Die voorkoms van oorgedraagde ARVMW is tans laag in vergelyking met geïndustrialiseerde lande, waarskynlik aangesien daar nog betreklik min tyd verloop het sedert k-ART wyd beskikbaar gemaak is. Volwassenes wat eerstelyn kombinasie terapie faal sal waarskynlik goed reageer op tweede-linie terapie, sonder terapie faling weens middelweerstandigheid. Daarenteen kan sommige kinders met protease inhibitor en tru-transkriptase weerstandigheid nie voldoende behandel word met die huidig-beskikbare middels in die uitrol program nie. Dit beklemtoon die dringende noodsaaklikheid van ‘n rasionele en wetenskaplike benadering tot k-ART in kinders, met ‘n lang terapie geskiedenis, wat toegang tot bykomende medikasie behels om `n derde-linie regimen saam te stel.
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Shao, Xingwu. "Reverse transcriptase assays for analysis of resistance to anti-HIV drugs and their mechanism of action /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-489-5.
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Bandaranayake, Rajintha M. "Exploring Molecular Mechanisms of Drug Resistance in HIV-1 Protease through Biochemical and Biophysical Studies: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/487.
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The human immunodeficiency virus type-1 (HIV-1) is the leading cause of acquired immunodeficiency syndrome (AIDS) in the world. As there is no cure currently available to treat HIV-1 infections or AIDS, the major focus of drug development efforts has been to target viral replication in an effort to slow down the progression of the infection to AIDS. The aspartyl protease of HIV-1 is an important component in the viral replication cycle and thus, has been an important anti-HIV-1 drug target. Currently there are nine protease inhibitors (PIs) that are being used successfully as a part of highly active antiretroviral therapy (HAART). However, as is with all HIV-1 drug targets, the emergence of drug resistance substitutions within protease is a major obstacle in the use of PIs. Understanding how amino acid substitutions within protease confer drug resistance is key to develop new PIs that are not influenced by resistance mutations. Thus, the primary focus of my dissertation research was to understand the molecular basis for drug resistance caused by some of these resistance substitutions.
Until recently, the genetic diversity of the HIV-1 genome was not considered to be important in formulating treatment strategies. However, as the prevalence of HIV-1 continues, the variability of the HIV-1 genome has now been identified as an important factor in how the virus spreads as well as how fast the infection progresses to AIDS. Clinical studies have also revealed that the pathway to protease inhibitor resistance can vary between HIV-1 clades. Therefore, in studying the molecular basis of drug resistance in HIV-1 protease, I have also attempted to understand how genetic variability in HIV-1 protease contributes to PI resistance.
In Chapters II, III and Appendix 1, I have examined how clade specific amino acid variations within HIV-1 CRF01_AE and clade C protease affect enzyme structure and activity. Furthermore, I have examined how these sequence variations, which are predominantly outside the active site, contribute to inhibitor resistance in comparison to clade B protease. With the results presented in Chapter II, I was able to show that sequence variations within CRF01_AE protease resulted in structural changes within the protease that might influence enzyme activity. In Chapter III, I focused on how sequence variations in CRF01_AE influence protease activity and inhibitor binding in comparison to clade B protease. Enzyme kinetics data showed that the CRF01-AE had reduced catalytic turnover rates when compared to clade B protease. Binding data also indicated that CRF01_AE protease had an inherent weaker affinity for the PIs nelfinavir (NFV) and darunavir (DRV). In work described in Chapter III, I have also examined the different pathways to NFV resistance seen in CRF01_AE and clade B protease. Using x-ray crystallographic studies I have shown the molecular mechanism by which the two different pathways confer NFV resistance. Furthermore, I provide a rational for why different resistance pathways might emerge in the two clades. In Appendix I, I present results from a parallel study carried out on clade C protease.
In Chapter IV, I have examined the role of residue 50 in HIV-1 protease in modulating inhibitor binding. Patients failing amprevavir (APV) and DRV therapy often develop the I50V substitution while the I50L substitution is often observed in patients failing atazanavir (ATV) therapy. This indicates that by making subtle changes at residue 50 the protease is able to confer differential PI resistance. With binding data presented in this chapter I have shown that substitutions at residue 50 change the susceptibility profiles of APV, DRV and ATV. Furthermore, from analyses of protease-inhibitor complexes, I have described structural insights into how substitutions at residue 50 can modulate inhibitor binding.
This thesis presents results that reveal mechanistic insights into how a number of resistance substitutions within protease confer drug resistance. The results on non-B clade proteases demonstrate that clade specific sequence variations play a role in modulating enzyme activity and influence the pathway taken to confer PI resistance. Furthermore, the results provide structural insights into how amino acid substitutions outside the active site effectively alter inhibitor binding.
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Cai, Yufeng. "Energetic and Dynamic Analysis of Inhibitor Binding to Drug-Resistant HIV-1 Proteases: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/448.
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HIV-1 protease is a very important drug target for AIDS therapy. Nine protease inhibitors have been proved by FDA and used in AIDS treatment. Due to the high replication rate and the lack of fidelity of the HIV-1 reverse transcriptase, HIV-1 virus developed various drug-resistant variants. Although experimental methods such as crystallography and isothermal titration calorimetry provide structural and thermodynamic data on drug-resistant variants, they are unable to discern the mechanism by which the mutations confer resistance to inhibitors. Understanding the drug-resistance mechanism is crucial for developing new inhibitors more tolerant to the drug-resistant mutations. Computational methods such as free energy calculations and molecular dynamic simulations can provide insights to the drug resistance mechanism at an atomic level. In this thesis, I have focused on the elucidation of the energetic and dynamics of key drug-resistant variants of HIV-1 protease.
Two multi-drug resistant variants, in comparison with wild-type HIV-1 protease were used for the comparisons: Flap+ (L10I, G48V, I54V, and V82A) which contains a combination of flap and active site mutations and ACT (V82T, I84V) that only contains active site mutations. In Chapter II, I applied free energy simulations and decomposition methods to study the differential mechanism of resistance to the two variants, Flap+ and ACT, to the recently FDA-approved protease inhibitor darunavir (DRV). In this study, the absolute and relative binding free energies of DRV with wild-type protease and the two protease variants were calculated with MM-PB/GBSA and thermodynamic integration methods, respectively. And the predicted results are in good agreement with the ITC experimental results. Free energy decomposition elucidates the mutations alter not only its own interaction with DRV but also other residues by changing the geometry of binding pocket. And the VdW interactions between the bis-THF group of DRV is predominant even in the drug-resistant variants. At the end of this chapter, I offer suggestions on developing new inhibitors that are based on DRV but might be less susceptible to drug-resistant mutations.
In Chapter III, 20-ns MD simulations of the apo wildtype protease and the apo drug-resistant protease variant Flap+ are analyzed and compared. In these studies, these mutations have been found to decrease the protease flexibility in the apo form but increase the mobility when the protease is binding with inhibitor.
In Chapter IV, more details of the free energy simulation and decomposition are discussed. NMR relaxation experiments were set up as a control for the MD simulation study of the dynamics of the Flap+ variant. The difficulty of finishing the NMR experiment is discussed and the solution and some preliminary results are shown.
In summary, the scope of this thesis was to use computational methods to study drug-resistant protease variants’ thermodynamic and dynamic properties to illuminate the mechanism of protease drug resistance. This knowledge will contribute to rational design of new protease inhibitors which bind more tightly to the protease and hinder the development of drug-resistant mutations.
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Abreu, Rodrigo Martins. "Avaliação dos desfechos virológicos e de adesão ao tratamento antiviral em pacientes portadores de hepatite B crônica." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/5/5168/tde-05102017-101708/.
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Introdução: A adesão ao tratamento da hepatite B crônica na vida real tem sido pouco estudada em todo o mundo. Neste estudo, foram avaliados os desfechos virológicos e de adesão ao tratamento antiviral de longo prazo em pacientes monoinfectados com hepatite B crônica. Métodos: Trata-se de um estudo prospectivo de coorte com pacientes portadores de hepatite B crônica (n = 183), tratados com adefovir, entecavir, lamivudina e / ou tenofovir, realizado em um centro de referência terciário brasileiro. A adesão ao tratamento foi avaliada por um questionário validado, denominado CEAT-HBV, em três momentos (2010/2011, 2013/2014 e 2014/2015). As variantes de resistência às drogas para hepatite B e a farmacocinética de um único ponto foram determinadas por sequenciamento e cromatografia líquida com espectrômetro de massa em tandem, respectivamente. Resultados: CEAT-HBV identificou 79/183 (43%) pacientes em não-adesão ao tratamento antiviral e entre esses, 53/79 (67%) tinham maior frequência de HBV DNA positiva. Porém, 38% (70/183) tiveram carga viral positiva sugerindo não resposta ao tratamento. As mais frequentes variantes de resistência aos antivirais foram M204I/V (78%), L180M (59%), L80I (15%), V173L (7%) e Q215H (6%). As principais causas associadas com a ausência de resposta ao tratamento antiviral foram variantes de resistência às drogas (39%), variantes de resistência às drogas e não adesão (23%), não adesão (13%), duração de tratamento insuficiente (10%), e indeterminada (16%). A farmacocinética de dose única indicou 48% (31/65) de não adesão ao antiviral. Dois anos depois da primeira avaliação, o CEAT-HBV indicou que 101/143 (71%) pacientes estavam em adesão ao tratamento, baseado na análise da população per-protocol. Entretanto, 21% (40/183) dos pacientes não puderam ser avaliados e foram excluídos. As principais razões para exclusão foram óbito (20/183), 11 dos 20 óbitos causados pelo carcinoma hepatocelular, perda de seguimento (16/183) e outras (4/183). Todos os participantes receberam nesse momento uma cartilha para orientação do tratamento. A terceira avaliação do CEAT-HBV (2014/2015) mostrou que 112/135 (83%) pacientes estavam em adesão ao tratamento (população per-protocol) e 8/143 (6%) foram excluídos. Desfechos de longo prazo mostraram que a taxa de adesão baseado no CEAT-HBV continua a aumentar após 4 anos (p < 0,001). Conclusões: Nossos dados realçam a importância do monitoramento da avaliação de adesão à terapia para hepatite B crônica. Desfechos de adesão de longo prazo podem ser dinâmicos e é possível aumentar a taxa de migração para o grupo com adesão/HBV DNA negativaBackground: Chronic hepatitis B (CHB) real-life treatment adherence has been poorly studied worldwide. In this study, it was evaluated long term virological and adherence outcomes regarding antiviral treatment in monoinfected CHB patients. Methods: A prospective cohort study with CHB patients (n=183) treated with adefovir, entecavir, lamivudine and / or tenofovir was performed in a Brazilian reference tertiary center. Treatment adherence was evaluated by a validate questionnaire named CEAT-HBV within three year-periods (2010/2011, 2013/2014 and 2014/2015). HBV drug resistance variants and single-dose pharmacokinetics were determined by sequencing and LC-MS/MS, respectively. Results: CEAT-HBV identified 79/183 (43%) patients with non-adherence to antiviral treatment and among them, 53/79 (67%) were more frequently viral load positive. However, 38% (70/183) had positive viral loads suggesting treatment non-response. Most frequent antiviral resistance variants were M204I/V (78%), L180M (59%), L80I (15%), V173L (7%) and Q215H (6%). The main causes associated with nonresponse to antiviral treatment were drug resistance variants (39%), drug resistance variants and nonadherence together (23%), non-adherence (13%), insufficient treatment duration (10%), and undetermined (16%). Single-dose pharmacokinetics indicated 48% (31/65) antiviral non-adherence. Two years after the first assessment, the CEATHBV indicated that 101/143 (71%) patients were adhered treatment, on basis of an analysis of the per-protocol population. However, 21% (40/183) of the patients could not be evaluated and were excluded. The main reasons for exclusion were death (20/183), 11 out 20 deaths due to hepatocellular carcinoma, loss to follow up (16/183) and others (4/183). HBV booklet was used for medical education. The third CEAT-HBV assessment (2014/2015) showed that 112/135 (83%) patients were on treatment adherence (per-protocol population) and 8/143 (6%) were excluded. Longterm evaluation showed that adherence rate based on CEAT-HBV continue to increase after 4-years (p < 0.001). Conclusions: Our data highlights the importance of CHB therapy adherence assessment monitoring. Long-term adherence outcomes may be dynamic and it is possible to increase the migration rate to adherence/HBV DNA negative group
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Bismara, Beatriz Aparecida Passos. "Padronização de tecnicas moleculares para o estudo da resistencia a drogas antiretrovirais em crianças infectadas pelo virus da imunodeficiencia humana tipo 1 (HIV-1) via perinatal." [s.n.], 2006. http://repositorio.unicamp.br/jspui/handle/REPOSIP/313491.
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Orientador: Sandra Cecilia Botelho CostaDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Investigamos a presença de mutações em crianças infectadas verticalmente pelo vírus da Imunodeficiência Humana tipo 1 (HIV-1) que conferem resistência aos agentes antiretrovirais. Amostras de sangue periférico foram coletadas de sessenta e seis pacientes em seguimento no do Ambulatório de Pediatria do Hospital das Clínicas da Universidade Estadual de Campinas. A partir de leucócitos destas amostras foi extraído o DNA, após diversas lavagens e precipitações. Foi preparado um Mix para 20 reações contendo 100µl de Buffer (50 mM de cloreto de potássio; 20 mM de Tris-HCl - pH 8,4); 100 µl de cloreto de magnésio (25mM); 12µl da mistura desoxirribonucléica - dNTPs (dATP, dGTP, dCTP; dTTP) a 25mM, 10µl de cada ¿primer¿ (25pmoles/µl); 0,5µl de Taq DNA polimerase e 0,5 µl do DNA a ser estudado, para a realização da PCR. As condições da reação foram: Desnaturação: 95ºC - 3 minutos; Anelamento: 55ºC ¿ 1 minuto; Extensão: 72ºC - 1 minuto (3 ciclos). Desnaturação: 95ºC ¿ 1 minuto; Anelamento: 55ºC ¿ 45 segundos; Extensão: 72ºC ¿ 1 minuto e Extensão final: 72ºC por 10 minutos (35 ciclos). As bandas foram visulizadas em gel de agarose 1%, obtendo-se uma banda de 1008 pares de bases. Os produtos selecionados foram submetidos a seguinte reação de seqüenciamento: 1,0 µl do produto da PCR; 4,0 µl de Premix (Amersham); 1,0 µl de primer 5 µM; Completar com dH2O para 10,0 µl de reação. Após a amplificação, os fragmentos foram analisados pelo Software MegaBACE¿ Sequence Analyzer, em seguida alinhados e comparados com o banco de dados público (GenBank), com o vírus selvagem, e com o programa Phred, Phrap, Consed. As principais mutações encontradas no gene da Transcriptase reversa foram: M184V(42,6%), M41L (39,3%), D67N (27,9%), T215Y (26,2%) e K70R (18%). No gene da Protease as principais encontradas foram: L63P (42,6%), L90M e M36I (32,8%), V77I (29,5%), I93L (24,6%), V82A (16,4%), I54V (14,8%) e K20R (13,1%). Concluímos que este método é muito eficaz para análise das seqüências, auxiliando na observação das mutações. Este exame deveria ser implantado na rotina para os pacientes portadores deste vírus, pois auxiliaria os clínicos na escolha de uma terapia ideal para cada paciente. As crianças que apresentavam estas mutações associadas às drogas antiretrovirais estavam com o quadro clínico afetado e a maioria dos esquemas terapêuticos não reduziam a carga viral em níveis desejáveis
Abstract: In this work our purpose was to determine the subtype, prevalence of drug-resistance mutations, and assess genotypic profiles in HIV-1 infected children under antiretroviral treatment, from Campinas, São Paulo, Brazil. Blood samples from sixty six vertically human immunodeficiency virus type 1 (HIV-1) infected Brazilian children were studied for antiretroviral drug resistance. Combination therapy with protease (PR) and reverse transcriptase (RT) inhibitors can efficiently supress human immunodeficiency virus (HIV) replication, but the emergence of drug resistance variants correlates strongly with therapeutic failure1. DNA was extracted from peripheral blood mononuclear cells (PBMc) samples, and a 1.0 kb fragment containing HIV-1PR and RT-coding sequence were amplified by Nested Polymerase Chain Reaction, sequencing and subtyping. The HIV-1 subtyping based on the polymerase (pol) gene sequences (protease and reverse transcriptase-RT regions) was as follow: subtype B (83,6%), subtype F (9,8%) and B/F viral recombinant forms (6,6%). We found fifty five sequences presented significant mutations and thirty five presented common polymorphism conferring resistance to protease inhibitors (PIs). Forty one presented mutations associated with resistant to nucleoside reverse transcriptase inhibitors (NRTIs). The entire viral protease and codons 1 to 219 of the reverse transcriptase gene from 61 HIV-1 isolates were amplified and sequenced for genotyping. Two major protease inhibitor-resistance associated mutations, M36I and L90M, were most prevalent in our samples (32,8%) and the polymorphism L69P (42,6%). Minor mutation were also found at the protease gene: V77I (29,5%), V82A (16,4%), I93L (24,6%), I54V (14,8%), K20R (13,1%). Many mutations associated with reduced susceptibility to nucleoside or non-nucleoside reverse transcriptase inhibitors were detected: M41L (39,3%), M184V (42,6%), D67N (27,9%), T215Y (26,2%), L210W (21%), K70R (18%) and E44D (11,5%). This study demonstrated that 98,3% of the studied population from the Campinas, showing evidences of therapy failure, presented viral genomic mutations associated with drug resistance. The main antiretroviral to which this population showed resistance were PI nelfinavir (54%), and ritonavir with lopinavir (18%), and the NNRTIs efavirenz (18%) and nevirapine (3,2%), and the NRTIs zidovudine (60,6%), didanosine (47,5%), lamivudine (45,9%). Ninety-eight percent of patients under antiretroviral therapy and with high viral load counts showed resistance to at least one of the antiretroviral analyzed.
Mestrado
Mestre em Farmacologia
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Cantrell, Ronald Alexander. "Diagnosing antiretroviral treatment failure in resource-limited settings." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008p/cantrell.pdf.
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Kalmar, Erika Maria do Nascimento. "Avaliação da resistência do HIV-1 às drogas anti-retrovirais em 150 pacientes em interrupção terapêutica por mais de seis meses." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/5/5134/tde-23102007-140855/.
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INTRODUÇÃO: A mudança nos critérios de introdução das drogas anti- retrovirais, assim como a dificuldade na manutenção da terapia anti-retroviral de alta eficácia, tem levado à descontinuação da terapêutica por longo período de tempo em alguns pacientes infectados pelo Vírus da Imunodeficiência Humana Adquirida-Tipo 1 (HIV-1). O objetivo deste estudo foi a caracterização dos fatores que levam à interrupção terapêutica e a avaliação da persistência da resistência aos anti-retrovirais após a interrupção da terapia anti-retroviral. MÉTODOS: Foram incluídos na pesquisa 150 pacientes de dois serviços de atendimento ambulatorial de atenção a pacientes infectados pelo HIV-1 da cidade de São Paulo, os quais se achavam em interrupção terapêutica havia pelo menos 6 meses. Os pacientes foram submetidos a um questionário e houve consulta aos prontuários. Foi realizada coleta de amostra de sangue para teste de genotipagem. O DNA pró-viral foi amplificado e seqüenciado para a região da protease e transcriptase reversa do vírus. As seqüências foram analisadas por meio do algoritmo de Stanford, sendo consideradas resistentes as amostras com resultado parcial ou completo de resistência a pelo menos uma droga. RESULTADOS: Dos 150 pacientes, 137 tiveram DNA do HIV-1 amplificado e seqüenciado, sendo que 38 (27,7%) apresentaram cepas resistentes. Entre os 38 pacientes com resistência, 29 (76,3%) apresentavam mutações para os análogos nucleosídeos inibidores da transcriptase reversa, 15 (39,4%) para os não análogos nucleosídeos inibidores da transcriptase reversa, e 5 (13,1%) para os inibidores da protease. A detectabilidade da carga viral antes da interrupção terapêutica foi o único fator associado com a resistência do vírus. Cento e dez (73,3%) pacientes suspenderam a medicação por orientação médica. A principal causa das interrupções terapêuticas foram os efeitos adversos para 58 (38,7%), seguida de 45 (30,0%) pacientes fora dos critérios atuais de início da terapia e/ou boas condições clínico/laboratoriais, e baixa adesão em 30 (20%). No ano anterior à pesquisa, 56 (37,3%) pacientes relataram relação sexual desprotegida e 130 (86,7%) mais que 2 parceiros. CONCLUSÕES: A freqüência de mutações de resistência revelou-se alta nesse grupo de pacientes. Tais mutações parecem ter um fitness semelhante ao das cepas selvagens, pois mesmo sem a pressão seletiva do medicamento por mais de 6 meses, mantiveram-se como cepas majoritárias. O aumento da carga viral, associado a comportamentos de risco, torna esses indivíduos uma fonte de cepas resistentes para a população, reforçando a necessidade de atenção especial para a prevenção da transmissão do HIV-1 nesse segmento de pacientes.INTRODUCTION: Changes in guidelines for antiretroviral introduction and difficulties in maintaining Highly Active Antiretroviral Therapy have lead some physicians in Brazil to interrupt for long periods of time the treatment in some Human Immunodeficiency Virus-1 (HIV-1) infected patients. The objective of this study was to evaluate the causes that influenced long term treatment interruption and to determine the frequency of resistant strains among these patients. METHODS: A total of 150 patients, previously treated with antiretroviral therapy and under treatment interruption TI for at least 6 months, were recruited from two HIV outpatients clinics in São Paulo city. Patients responded to a questionnaire and the medical records were also analyzed. Plasma samples were obtained to HIV-1 genotypic resistance test. DNA was amplified for the protease and reverse transcriptase gene. Sequences were analyzed using Stanford algorithm; samples were considered resistant if they resulted in partial or complete resistance to at least one drug. RESULTS: One hundred thirty seven of the 150 samples had their DNA amplified, 38 (27.7%) of them harboring a resistant strain. Nucleoside reverse-transcripatse inhibitors, nonnucleoside reverse- transcripatse inhibitors and protease inhibitors associated mutations were present in 29 (76.3), 15 (39.4%) and 5 (13.1%) samples respectively. We could only associate presence of resistance to viral load detection before TI.. Of the 150 patients, 110 (73.3%) had interrupted treatment following medical advice, the remaining stopped by their own decision. The reasons for TI were: 58 (38.7%) had ARV-related side-effects, 45 (30.0%) had good laboratory parameter and/or started therapy based on criteria that were no longer used, 30 (20.0 %) had poor adhesion. During the 12 months prior to the study, there were 56 (37.3%) who had unprotected sexual relations and 130 (86.7%) had had sex with two or more partners. CONCLUSION: The frequency of drug resistance strains in this group of patients was high. These strains seem to have a good fitness because they were present after 6 months of drug interruption. The high viral load associated to non sexual protection in this group of patients may lead to increase in transmission of drug resistance strains.This highlights the need of prevention measures in this special group.
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Colomer, Lluch Marta. "Antibiotic resistance genes in the viral DNA fraction of environmental samples = Gens de resistència a antibiòtics en el DNA de la fracció vírica de mostres ambientals." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/144525.
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The PhD thesis presented here has as a main objective the study of antibiotic resistance genes clinically relevant in the DNA fraction of bacteriophage particles isolated from environmental samples of different origin in order to determine the importance of bacteriophages as vehicles for the mobilization of antibiotic resistance genes between bacteria.
Specifically, a broad range of antibiotic resistance genes were studied as representative of the main groups recently described in our geographical area belonging to three β-lactamases (blaTEM, blaCTXM-1 and blaCTX-M-9), the mecA gene conferring resistance to methicillin in staphylococci, and the quinolones resistance genes qnrA and qnrS.
To achieve these goals samples of urban wastewater, river water and animal faecal wastes were analysed quantifying the antibiotic resistance genes of interest in bacteriophages DNA.
During the development of this Thesis, it was attempted to optimize the available methodology for bacteriophage DNA extraction, as well as the necessary controls to guarantee the amplification of encapsidated DNA and to remove any free DNA in the samples and any possible vesicles containing DNA.
In addition, the ability of phage-encoded genes to confer antibiotic resistance in bacterial strains was assessed by performing transformation experiments.
It was also studied the influence of various compounds involved in the induction of the lytic cycle of temperate bacteriophages, on the abundance of antibiotic resistance genes in DNA from the phage fraction in wastewater samples.
Finally, due to the importance of horizontal gene transfer as a mechanism for antibiotic resistance dissemination in clinical and environmental settings, transduction experiments were attempted to reproduce in vitro the process that would take place in nature.
The research developed in this Thesis is divided into 5 studies included in 4 chapters: (1) Antibiotic resistance genes in the bacteriophage DNA fraction of water samples (wastewater, river water and animal wastewater); (2) Quinolone resistance genes (qnrA and qnrS) in bacteriophage particles from wastewater samples and the effect of inducing agents on packaged antibiotic resistance genes; (3) Evaluation of ARGs in the DNA of bacterial and bacteriophage fraction in wastewater samples from Tunisia and comparison with results obtained in Barcelona area; (4) Detection of quinolone-resistant Escherichia coli isolates belonging to clonal groups O25b:H4-B2-ST131 and O25b:H4-D-ST69 in water samples from Barcelona area.
Each of the studies has given rise to a scientific article already published or submitted for scientific publication.La tesi doctoral que es presenta a continuació té com a objectiu principal l’estudi de gens de resistència a antibiòtics de rellevància clínica en la fracció de DNA de partícules de bacteriòfags aïllades de diferents tipus de mostres ambientals per tal de determinar la importància dels bacteriòfags com a vehicles de mobilització de gens de resistència a antibiòtics entre bacteris. S’ha estudiat un ampli espectre de gens de resistència a antibiòtics com a representants dels grups principals descrits actualment en la nostra àrea geogràfica corresponent a tres β-lactamases (blaTEM, blaCTXM-1 i blaCTX-M-9), el gen mecA de resistència a meticil•lina en estafilococs, i els gens de resistència a quinolones qnrA i qnrS. Per això s’han analitzat diversos tipus de mostres procedents d’aigua residual municipal, d’aigua de riu i d’aigua residual amb contaminació fecal animal per tal de quantificar els gens de resistència a antibiòtics d’interès en DNA aïllat de bacteriòfags. Durant els diferents estudis s’ha intentat optimitzar la metodologia d’extracció de DNA de bacteriòfags així com els controls corresponents per garantir l’amplificació de DNA encapsidat i l’eliminació de qualsevol DNA lliure present a les mostres i de qualsevol possible vesícula amb DNA al seu interior. Per altra banda, també s’ha determinat la capacitat funcional dels gens de resistència detectats en DNA de fags i per això s’han realitzat experiments de transformació a partir de soques bacterianes sensibles a un determinat antibiòtic amb l’objectiu d’incorporar la resistència i per tant, esdevenir resistents a l’antibiòtic en qüestió. També, s’ha estudiat la influència de determinats compostos implicats en la inducció del cicle lític de bacteriòfags temperats, en l’augment en el nombre de còpies de gens de resistència a antibiòtics en DNA present en la fracció de fags de l’aigua residual. Finalment, degut a la importància de la transferència horitzontal de gens com a mecanisme de dispersió de la resistència a antibiòtics en el medi ambient i en clínica s’han dut a terme experiments de transducció per tal d’intentar reproduir in vitro el procés que tindria lloc de manera natural.
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Lange, C. M. "Population and single genome kinetics driving the evolution of multiple linked multiclass drug resistance mutations in the viral protease and reverse transcriptase of HIV-1 subtype C in children receiving early protease inhibitor based combination therapy." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1469608/.
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This thesis examines the evolution of HIV-1 subtype C multiple linked multi-class antiretroviral resistance mutations in the viral protease (PR) and reverse transcriptase (RT) genes of vertically infected children. Emergence of PI resistance on the backdrop of pre-existing non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance could compromise long-term treatment options in such children. We characterised multi-class drug resistance using single genome sequencing (SGS) in children with viraemia while receiving PI-based ART. We applied SGS of HIV-1 protease (PR) and reverse transcriptase (RT) to longitudinal samples from a cohort of the Children with HIV Early Antiretroviral Therapy (CHER) trial with viral loads >1000c/ml after 40 weeks of early ART. Bulk sequencing revealed NVP-selected resistance in 50% of these children while SGS revealed NVP-selected resistance in 70%. Two children had baseline NRTI and PI mutations, suggesting previous maternal ART. Linked multi-class drug resistance following PI-based ART was detected by SGS in 2/10 children. In one child, the majority species contained M184V in RT linked to L10F, M46I/L, I54V and V82A in PR and a triple-class drug resistant variant with these mutations linked to the NNRTI mutation V108I. In the second child, the majority species contained M184V and V82A linked within viral genomes. I correlated nucleotide variation of PR-RT with the number of single genomes obtained at each time point and ART status and used maximum likelihood trees, recombination analysis, positive selection analysis and co-evolution analysis to describe the evolution of PR-RT of the viral populations. Six children who received early ART for 40/96 weeks only or received continuous ART for the duration of the CHER trial had clusters of identical sequences from baseline and week 40 of ART. These sequences did not harbour known drug resistance mutations. Therefore one could hypothesize viral replication from a persisting viral reservoir that was established from infection that occurred prior to the initiation of ART. The rooted ML trees of 2 children who developed drug resistance during ART had clusters of identical sequences harbouring common drug resistance mutations from multiple time points which is characteristic of the selection of drug resistant viral populations that cause virological failure during ART. When drug resistant viral populations developed during treatment failure, M184V single mutated viruses were selected from multiple wildtype viral populations but only one population became the major contributor to drug resistant viraemia in both children. Triple-class drug resistant sequences that had common DRMs (M184V, V108I in RT and M46I in PR) did not cluster together. I found no evidence of recombination or coevolving sites in PR-RT for any of these children. I used a luciferase based single replication cycle assay to examine drug susceptibility and replication capacity (RC) conferred by multi-class drug resistant PR-RT from the 2 children who developed such drug resistant variants. I tested the susceptibility of pseudoviruses to the components of early ART (AZT, 3TC and LPV), the components of second-line therapy for these children (Abacavir (ABC), Didanosine (ddI), Efavirenz (EFV) and (NVP)), the PIs Nelfinavir (NFV) and Saquinavir (SQV), which are also approved for use in children and Darunavir (DRV), which has been identified as a PI option needed in paediatric co-formulation. Pseudoviruses with known PI resistance conferring mutations showed reduced susceptibility to all PIs except DRV. Those with known NNRTI resistance conferring mutations showed reduced susceptibility to EFV and NVP. M184V mutated pseudoviruses conferred high-level resistance to 3TC. In one child, a combination or one of the RT mutations V35T, E36D, T39R, S48T, T165I, K173A, D177E, T200A, Q207D, R211K, V245Q, E248N, D250N, A272P, K277R, E291D, I293V, T296N may be associated with high-level ABC and ddI resistance when genetically linked with M184V. Population sequence analysis was used to characterize the viral gag genes that encoded matrix, capsid, nucleocapsid, p6, and spacer peptides 1 and 2 along with PR-RT as a single amplicon. I determined the presence of compensatory PI-resistance mutations in gag, drug resistance mutations in PR and RT and other amino acid changes that occurred during ART. To determining the polymorphic nature of these sites, I compared them to a position-specific scoring matrix for gag that was derived from HIV-1 subtype C sequences from children from Sub-Saharan Africa. P453L in the p1/p6 cleavage site of Gag emerged in the viral population of one child during PI-based ART. It was the only amino acid change in Gag that emerged among all children in the study cohort that has been characterised as a compensatory mutation that is selected by and enhances PI-resistance. This project is the first to identify multi-class drug resistance mutations in PR and RT that were linked on the same genome as well as characterise their development during early PI-based ART in children. Triple class drug resistant viruses detected in the minority species of the viral population of one child demonstrated significant levels of resistance to LPV, SQV, NFV, 3TC and NVP, and established that such variants could compromise future ART regimes if they became the dominant species of the viral population. I note that the small convenience sample (n = 10) chosen for this project limited the power of this study so that findings could not be generalized.
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Kolli, Madhavi. "Co-evolution of HIV-1 Protease and its Substrates: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/455.
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Drug resistance is the most important factor that influences the successful treatment of individuals infected with the human immunodeficiency virus type 1 (HIV-1), the causative organism of the acquired immunodeficiency syndrome (AIDS). Tremendous advances in our understanding of HIV and AIDS have led to the development of Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, to combat the virus. Though HAART has been successful in reducing AIDS-related morbidity and mortality, HIV rapidly evolves resistance leading to therapy failure. Thus, a better understanding of the mechanisms of resistance will lead to improved drugs and treatment regimens.
Protease inhibitors (PIs) play an important role in anti-retroviral therapy. The development of resistance mutations within the active site of the protease greatly reduces its affinity for the protease inhibitors. Frequently, these mutations reduce catalytic efficiency of the protease leading to an overall reduction in viral fitness. In order to overcome this loss in fitness the virus evolves compensatory mutations within the protease cleavage sites that allow the protease to continue to recognize and cleave its substrates while lowering affinity for the PIs. Improved knowledge of this substrate co-evolution would help better understand how HIV-1 evolves resistance and thus, lead to improved therapeutic strategies. Sequence analyses and structural studies were performed to investigate co-evolution of HIV-1 protease and its cleavage sites. Though a few studies reported the co-evolution within Gag, including the protease cleavage sites, a more extensive study was lacking, especially as drug resistance was becoming increasingly severe.
In Chapter II, a small set of viral sequences from infected individuals were analyzed for mutations within the Gag cleavage sites that co-occurred with primary drug resistance mutations within the protease. These studies revealed that mutations within the p1p6 cleavage site coevolved with the nelfinavir-resistant protease mutations. As a result of increasing number of infected individuals being treated with PIs leading to the accumulation of PI resistant protease mutations, and with increasing efforts at genotypic and phenotypic resistance testing, access to a larger database of resistance information has been made possible. Thus in Chapter III, over 39,000 sequences were analyzed for mutations within NC-p1, p1-6, Autoproteolysis, and PR-RT cleavage sites and several instances of substrate co-evolution were identified. Mutations in both the NC-p1 and the p1-p6 cleavage sites were associated with at least one, if not more, primary resistance mutations in the protease.
Previous studies have demonstrated that mutations within the Gag cleavage sites enhance viral fitness and/or resistance when they occur in combination with primary drug resistance mutations within the protease. In Chapter III viral fitness in the presence and absence of cleavage site mutations in combination with primary drug resistant protease mutations was analyzed to investigate the impact of the observed co-evolution. These studies showed no significant changes in viral fitness. Additionally in Chapter III, the impact of these correlating mutations on phenotypic susceptibilities to various PIs was also analyzed. Phenotypic susceptibilities to various PIs were altered significantly when cleavage site mutations occurred in combination with primary protease mutations. In order to probe the underlying mechanisms for substrate co-evolution, in Chapter IV, X-ray crystallographic studies were performed to investigate structural changes in complexes of WT and D30N/N88D protease variants and the p1p6 peptide variants. Peptide variants corresponding to p1p6 cleavage site were designed, and included mutations observed in combination with the D30N/N88D protease mutation. Structural analyses of these complexes revealed several correlating changes in van der Waals contacts and hydrogen bonding as a result of the mutations. These changes in interactions suggest a mechanism for improving viral fitness as a result of co-evolution.
This thesis research successfully identified several instance of co-evolution between primary drug resistant mutations in the protease and mutations within NC-p1 and p1p6 cleavage sites. Additionally, phenotypic susceptibilities to various PIs were significantly altered as a result of these correlated mutations. The structural studies also provided insights into the mechanism underlying substrate co-evolution. These data advance our understanding of substrate co-evolution and drug resistance, and will facilitate future studies to improve therapeutic strategies.
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Brice, Joséphine. "Caractérisation du réservoir viral et des anticorps chez des enfants infectés par le VIH en suppression virologique au Mali." Electronic Thesis or Diss., Sorbonne université, 2019. http://www.theses.fr/2019SORUS506.
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L’absence de virémie VIH détectable chez 3 enfants après l’arrêt du traitement antirétroviral suggère qu’une thérapie très précoce pourrait amener à une cure fonctionnelle. L’objectif principal est de caractériser de manière qualitative et quantitative le réservoir viral ainsi que les anticorps anti-VIH chez des enfants en suppression virologique afin d’identifier les facteurs associés à leur diminution. Il s’agit d’une étude prospective transversale incluant 97 enfants, au CHU Gabriel Touré (Bamako, Mali). Ils avaient un âge médian de 9,8 ans et de 3,3 ans à la mise en route du traitement avec une durée médiane de traitement de 5,4 années. Le taux médian d’ADN-VIH était de 445 copies/106 PBMCs, la médiane d’activité des anticorps anti-gp41 était de 0,29 DO et le niveau médian des anticorps anti-VIH était 14,1 S/CO. Nous avons montré une prévalence élevée de la résistance au VIH dans l'ADN. Huit séroréversions VIH ont été identifiées. Une activité basse des anticorps anti-gp41 était associée à un plus jeune âge d’initiation du traitement et avec un faible niveau d’anticorps anti-VIH. Un faible niveau d’anticorps anti-VIH était associée à une initiation précoce du traitement. Une proportion significative d'enfants VHIV en suppression virologique ayant commencé une thérapie avant l'âge de 2 ans ont cessé de produire et/ou ont progressivement perdu les anticorps anti-VIHAbsence of detectable HIV viremia treatment cessation in 3 children suggests that very early could lead to functional cure. The main objective is to qualitatively and quantitatively characterize viral reservoir and anti-HIV-1 antibodies virologically suppressed children in order to identify factors associated with their decrease. This is a prospective cross-sectional study included 97 children at the Gabriel Touré University Hospital (Bamako, Mali). They had a median age of 9.8 years at time of inclusion and 3.3 years at treatment initiation with a median HAART duration of 5.4 years. The median total HIV-DNA level was 445 copies/106 PBMCs, the median anti-gp41 antibodies activity was 0.29 optical density and the median HIV antibody level was 14.1 S/CO. We showed a high prevalence of HIV-1 resistance in HIV-DNA. Eight seroreversions were identified. A low anti-gp41 antibody activity was associated with both a younger age at HAART initiation and a lower level of anti-HIV antibodies. A lower anti-HIV antibodies level was associated with a younger age at HAART initiation. A significant proportion of virologically suppressed VHIV children who initiated HAART before the age of 2 years stopped to produce and/or progressively lost the HIV antibodies
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Mittal, Seema. "Role of Protein Flexibility in Function, Resistance Pathways and Substrate Recognition Specificity in HIV-1 Protease: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/573.
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In the 30 years since the Center for Disease Control's Morbidity and Mortality Weekly Report published the first mention of what later was determined to be AIDS (Acquired immunodeficiency syndrome) and HIV (Human immunodeficiency virus) recognized as the causative pathogen, much has been done to understand this disease’s pathogenesis, development of drugs and emergence of drug resistance under selective drug therapy. Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, have helped stabilize the HIV prevalence at extraordinarily high levels. Despite the recent stabilization of this global epidemic, its dimensions remain staggering with estimated (33-36 million) people living with HIV-AIDS in 2007 alone. This is because the available drugs against AIDS provide treatment for infected individuals, but HIV evolves rapidly under drug pressure and develops resistant strains, rendering the therapy ineffective. Therefore, a better understanding underlying the molecular mechanisms of viral infection and evolution is required to tackle drug resistance and develop improved drugs and treatment regimens.
HIV-1 protease is an important target for developing anti-HIV drugs. However, resistant mutations rapidly emerge within the active site of the protease and greatly reduce its affinity for the protease inhibitors. Frequently, these active site drug resistant mutations co-occur with secondary/ non-active site/ associated or compensatory mutations distal to the active site. The role of these accessory mutations is often suggested to be in maintaining viral fitness and stability of protease. Many of the non-active site drug resistant mutations are clustered in the hydrophobic core in each monomer of the protease. Molecular dynamic simulation studies suggest that the hydrophobic core residues facilitate the conformational changes that occur in protease upon ligand binding. There is a complex interdependence and interplay between the inherent adaptability, drug resistant mutations and substrate recognition by the protease. Protease is inherently dynamic and has wide substrate specificity. The PI (protease inhibitor) resistant mutations, perhaps, modulate this dynamics and bring about changes in molecular recognition, such that, in resistant proteases, the substrates are recognized specifically over the PIs for the same binding site. In this thesis research, I have investigated these three complementary phenomena in concert.
Chapter II examines the importance of hydrophobic core dynamics in modulating protease function. The hydrophobic core in the WT protease is intrinsically flexible and undergoes conformational changes required for protease to bind its substrates. This study investigated if dynamics is important for protease function by engineering restricted vs. flexible hydrophobic core region in each monomer of the protease, using disulfide chemistry. Under oxidizing conditions, disulfide bond established cross-link at the interface of putative moving domains in each monomer, thereby, restricting motion in this region. Upon reduction of the disulfide bond, the constraining influence was reversed and flexibility returned to near WT. The disulfide cross-linked protease showed significant loss of function when tested in functional cleavage assay. Two protease variants (G16C/L38C) and (R14C/E65C) were engineered and examined for changes in structure and enzymatic activity under oxidizing and reducing conditions. (R14C/E65C) was engineered as an internal control variant, such that cysteines were engineered between putative non-moving domains. Structurally, both the variants were very similar with no structural perturbations under oxidizing or reducing conditions. While significant loss in function was observed for (G16C/L38C) only under oxidizing conditions, (R14C/E65C) did not show any loss of function under oxidizing or reduced conditions, as expected. Successful regain of function for cross-linked (G16C/L38C) was obtained upon reversible reduction of the disulfide bond. Taken together, these data demonstrate that the hydrophobic core dynamics modulates protease function and support the hypothesis that the distal drug resistant mutations, possibly causing drug resistance by modulating hydrophobic core dynamics via long range structural perturbations. Since protease recognizes and cleaves more than 10 substrates at different rates, our further interest is to investigate if there is a differential loss of activity for some specific substrates over the others, and whether the order of polypeptide cleavage is somehow affected by restricted core mobility. In order to better answer these questions it is essential to understand: what determines the substrate binding specificity in protease? A two-pronged approach was applied to address this question as described in chapter III and IV respectively.
In chapter III, I investigated the determinants of substrate specificity in HIV-1 protease by using computational positive design and engineered specificity-designed asymmetric protease (Pr3, A28S/D30F/G48R) that would preferentially bind to one of its natural substrates, RT-RH over two other substrates, p2-NC and CA-p2, respectively. The designed protease was expressed, purified and analyzed for changes in structure and function relative to WT. Kinetic studies on Pr3 showed that the specificity of Pr3 for RT-RH was increased significantly compared to the wild-type (WT), as predicted by the positive design. ITC (Isothermal Titration Calorimetry) studies confirmed the kinetic data on RT-RH. Crystal structural of substrate complexes of WT protease and Pr3 variant with RT-RH, CA-p2 and p2-NC were further obtained and analyzed. The structural analysis, however, only partially confirmed to the positive design due to the inherent structural pliability of the protease. Overall, this study supports the positive computational design approach as an invaluable tool in facilitating our understanding of complex proteins such as HIV 1 protease and also proposes the integration of internal protein flexibility in the design algorithms to make the in-silico designs more robust and dependable.
Chapter IV probed the substrate specificity determining factors in HIV-1protease system by focusing on the substrate sequences. Previous studies have demonstrated that three N-terminal residues immediate to the scissile bond (P1-P3) are important in determining recognition specificity. This work investigated the structural basis of substrate binding to the protease. Catalytically active WT protease was crystallized with decameric polypeptides corresponding to five of the natural cleavage sites of protease. The structural analyses of these complexes revealed distinct P side product bound in all the structures, demonstrating the higher binding affinity of N terminal substrate for protease.
This thesis research successfully establishes that intrinsic hydrophobic core flexibility modulates function in HIV-1 protease and proposes a potential mechanism to explain the role of non-active site mutations in conferring drug resistance in protease. Additionally, the work on specificity designed and N terminal product bound protease complexes advances our understanding of substrate recognition in HIV protease.
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Neto, Gaspar Lisbôa. "Identificação de polimorfismos e mutações primárias de resistência aos inibidores de protease (NS3/NS4A) no vírus da hepatite C em pacientes com hepatite C crônica monoinfectados e coinfectados pelo vírus da imunodeficiência humana." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/5/5134/tde-31072017-150758/.
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INTRODUÇÃO: A hepatite C crônica é uma das principais causas de hepatopatia em todo mundo. A coinfecção pelo vírus C (VHC) e o HIV não é incomum, pois ambos compartilham vias similares de transmissão. Recentemente, a terapêutica da hepatite C crônica foi radicalmente modificada com o advento das drogas antivirais de ação direta (DAAs), elevando as taxas de RVS mesmo na população coinfectada. O VHC é caracterizado pela sua alta taxa replicativa e por grande diversidade populacional. Substituições de ocorrência natural na protease viral associadas a resistência podem comprometer a terapêutica em alguns regimes baseados no uso de inibidores de protease (IPs). OBJETIVOS: Estimar a prevalência de polimorfismos e mutações de ocorrência natural associadas a resistência aos IPs em pacientes monoinfectados e coinfectados pelo VHC e HIV e identificar fatores clínicos e virológicos associados a presença de tais substituições. MATERIAIS E MÉTODOS: Dados epidemiológicos e clínicos foram obtidos de 247 pacientes (135 monoinfectados e 112 coinfectados pelo VHC e HIV). VHC RNA foi extraído do plasma dos indivíduos participantes e um fragmento de 765 pares de base da região NS3 foi amplificado e sequenciado por metodologia populacional (técnica de Sanger). O estadiamento da fibrose hepática foi realizado pelo escore não invasivo FIB- 4. RESULTADOS: 54 indivíduos (21,9%) apresentaram pelo menos uma substituição na região NS3/NS4A do VHC. Somente 14 pacientes (5,7%) apresentaram pelo menos uma mutação de resistência aos IPs (T54S, V55A ou Q80R). A Q80K não foi identificada em nenhuma das amostras. Não houve diferença entre monoinfectados e coinfectados quanto à ocorrência de polimorfismos ou mutações associadas a resistência. As variáveis independentemente associadas com substituições na região da protease foram infecção pelo VHC genótipo 1b, bilirrubinas totais > 1,5 vezes o LSN e níveis de albumina < 3,5 g/dL. Fibrose hepática avançada (FIB-4 > 3.25) não esteve associada a presença de substituições. A análise de diversidade nucleotídica na protease viral revelou maior heterogeneidade do VHC genótipo 1b em relação ao 1a. Contudo, a análise de pressão seletiva não demonstrou maior variabilidade de quasiespécies no grupo de hepatopatia avançada, achado este compatível com uma sequência genômica relativamente conservada. CONCLUSÕES: As substituições na região NS3/NS4 do VHC consistiram majoritariamente por polimorfismos naturais sem impacto clínico num eventual tratamento que envolva o uso de IPs. A prevalência de substituições associadas a resistência foi baixa e compatível com os valores informados pela maioria dos estudos nacionais e internacionais. A coinfecção pelo HIV não parece elevar a frequência de substituições na protease do VHC. A região NS3 do genótipo 1b foi altamente variável em relação ao genótipo 1a, reforçando o conceito de possíveis diferenças geográficas em relação ao perfil genético deste vírusINTRODUCTION: Chronic hepatitis C is a major cause of liver disease worldwide. Hepatitis C vírus (HCV) and HIV coinfection is not uncommon due to similar transmission routes. Recently developed direct-acting antivirals drugs (DAAs) have increased the rate of SVR even in coinfected patients. HCV has a high replication rate and a lack of proofreading activity, leading to a greatly diverse viral population. Baseline spontaneously occurring resistance substitutions in the protease region may impair the rate of success in some protease inhibitors (PI) based regimens. OBJECTIVE: to determine the prevalence of naturally occurring polymorphisms and resistance associated variants to HCV PIs in mono and coinfected HCV HIV patients and to evaluate potential associations between amino acid substitutions in protease domain and clinical / virological features of those patients. METHODS: Clinical and epidemiological data were retrieved from medical records of 247 subjects in Brazil (135 HCV monoinfected and 112 HIV HCV coinfected patients). HCV-RNA was extracted from plasma and a fragment of 765 base pairs from the NS3 region was amplified and sequenced with Sanger-based technology. Fibrosis staging was assessed by non invasive score (FIB-4). RESULTS: Overall, 54 patients (21.9%) had at least one amino acid substitution in the NS3 region; only 14 patients (5.7%) harboured at least one resistance mutation (T54S, V55A, Q80R). Q80K mutation was not found in any sample. There was no difference between monoinfected and coinfected patients regarding the frequency of natural polymorphisms and resistance mutations. Variables independently associated with amino acid substitution were HCV subtype 1b, total bilirubin level > 1.5 ULN and albumin level < 3.5 g/dL. Advanced liver fibrosis (FIB-4 > 3.25) was not related to NS3 polymorphisms nor resistance associated variants. Examination of HCV protease nucleotide diversity revealed greater heterogeneity in subtype 1b than subtype 1a. Analysis of selective pressure did not reveal a greater quasispecies variability in advanced liver fibrosis group, being such finding consistent with a relatively conserved gene in this setting. CONCLUSION: Baseline HCV NS3 amino acid substitutions depicted herein were considered mostly natural polymorphisms with no clinical impact in a PI based therapy. The prevalence of resistance-associated substitutions was low and compatible with values reported by most national and international studies. HIV coinfection was not associated with a greater frequency of such substitutions in the studied sample. The NS3 region of genotype 1b was highly variable in relation to genotype 1a, highlighting geographic differences concerning HCV genetic profile
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Soares, Celina Maria Pereira de Moraes [UNIFESP]. "Prevalência de resistência transmitida do HIV-1 aos antirretrovirais no Brasil, pré- início de tratamento." Universidade Federal de São Paulo (UNIFESP), 2011. http://repositorio.unifesp.br/handle/11600/9962.
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Previous issue date: 2011-09-28A seleção de mutações de resistência aos medicamentos antirretrovirais pós-falha terapêutica representam um grande desafio para a tomada de decisão de novos esquemas de tratamento na pandemia global. A elevada variabilidade genética do HIV-1 e a seleção de mutações de resistência transmitida a pacientes infectados cronicamente, sem que tenham participado de qualquer esquema terapêutico, tem sido objeto de vários estudos no mundo. Os padrões de resistência são estudados principalmente em países da Europa e Estados Unidos, que apresentam prevalência majoritária do subtipo B. Contudo, estudos que direcionam a seleção de mutações transmitidas aos medicamentos antirretrovirais e em subtipos não-B, assim como em suas formas recombinantes, tem aumentado significativamente em várias regiões do mundo. No Brasil, esses estudos são realizados esporadicamente e em regiões distintas do país e, principalmente, em pacientes recém-infectados. A alta variabilidade genética do HIV-1 no nosso país é representada de forma diversificada, com a presença do subtipo B, seguida do F, e especificamente na região Sul a importante prevalência do subtipo C. O objetivo principal deste estudo está embasado nas características de mutações de resistência transmitida aos medicamentos antirretrovirais pelo HIV-1 no gene pol, frações da transcriptase reversa e protease, com análise do perfil mutacional por grupos populacionais de pacientes cronicamente infectados pelo HIV-1 e não tratados, porém com indicação de início imediato de tratamento. Foram avaliados os pacientes representados nas regiões demográficas do Brasil. A prevalência nacional, resultou em 12,1% de mutações de resistência transmitida aos antirretrovirais pelo HIV-1 (grau intermediário de 5 a 15%) e 70,8% de subtipo B; 15,5% C; 6,4% F; 4,0% BF e 3,0% BC na classificação dos subtipos do HIV-1. Além disso, foram classificadas as prevalências de mutações transmitidas, dos subtipos do HIV-1 e características sociodemográficas, laboratoriais e os dados comportamentais na população HIV positiva pré-terapia por cidade nas cinco regiões brasileiras.
The selection of resistance mutations to antiretroviral drugs after failure of antiretroviral therapy represents a major challenge for decision-making of new therapeutic regimens in the global pandemic. The high genetic variability of HIV- 1and the selection of resistance mutations trasmitted to patients chronically infected without having participated in the regimen has been the subject of several studies in the world. Resistance patterns are studied mainly in European countries and the United States, wich have majority prevalence of subtype B. However, studies that guide the selection of transmitted mutants to antiretroviral drugs and non-B subtypes, and in their recombinant forms, has increased significantly in the several regions of the world. In Brazil, these studies are conducted sporadically and in different regions of the country and specially in newly infected patients. The high genetic variability of HIV-1 is represented in our country so diverse, with the presence of subtype B, followed by F, and specifically in the South region, the prevalence of subtype C. In addition, coexist the prevalence of recombinant forms, where the principal is the subtype BF, followed by BC. The main objective of this study estimate the characteristics of transmitted resistance mutations to antiretroviral drugs in HIV-1 ol gene, fractions of reverse transcriptase and protease, with mutational analysis of the profile by a population patients chronically infected with HIV-1 and not treated, but with indication of immediate initiation of treatment. We evaluated the patients represented in the demographic regions of Brazil. The national prevalence resulted in 12.1% of transmitted resistance mutations to antiretroviral (intermediate grade 5% to 15%) and 70.8%, 15.5% C, 6.4% F, 4.0% BF and 3.0% BC in the classification of subtypes of HIV-1. In addition, the prevalence of transmitted mutations, the subtypes of HIV-1 and sociodemographic characteristics, laboratory parameters and behavior data in population HIV-1 positive pre-treatment were classified by the cities in five Brazilian regions.
TEDE
BV UNIFESP: Teses e dissertações
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Franke, Christina E. "Tobacco Mosaic Virus Nanocarrier for Restored Cisplatin Efficacy in Platinum-Resistant Ovarian Cancer." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1493810190306879.
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Diouara, Abou Abdallah Malick. "Réponse virologique au traitement antirétroviral chez les patients infectés par le VIH-1, suivis en milieux décentralisés en Afrique de l’Ouest (Sénégal, Mali et Guinée Conakry)." Thesis, Montpellier 1, 2014. http://www.theses.fr/2014MON1T013/document.
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L'une des principales barrières à la prise en charge optimale des patients sous traitement antirétroviral est l'accès limité aux tests de charge virale (CV) et de génotypage particulièrement en milieu décentralisé. Ces tests ne sont généralement disponibles qu'au niveau des structures sanitaires centrales de grandes villes et le plasma en est l'échantillon de référence. Or, son transfert des régions périphériques vers les laboratoires de références est difficile, voire impossible. Pour rapprocher les patients du laboratoire, nous avons démontré la possibilité d'assurer un suivi virologique complet (CV et génotypage) à partir des DBS collectés et acheminés dans des conditions de terrain. Nous avons également pour la première fois, documenté la réponse virologique au traitement antirétroviral et la diversité génétique du VIH-1 chez des patients adultes suivis en milieux décentralisés au Sénégal, au Mali et en Guinée Conakry. Globalement, malgré les défauts d'observance au traitement souligné, les résultats de nos travaux ne montrent pas de différences significatives dans la survenue de l'échec virologique entre patients suivis dans les structures sanitaires centrales et périphériques, ceci quelque soit le pays considéré. Au Sénégal, chez les enfants nés de mères séropositives, la résistance vis à vis des INNTI était plus prépondérante, probablement du fait de l'utilisation systématique de la Névirapine durant la PTME. Par ailleurs, aucune mutation de résistance aux inhibiteurs d'intégrase n'a été observée malgré des taux de résistance élevés chez des patients en échec de première et deuxième ligne de traitement. Nos travaux confirment également une grande diversité génétique des sous-types viraux avec cependant la prédominance du CRF02_AG dans la sous région Ouest Africaine. Ces travaux de thèse mettent en évidence la faisabilité et la pertinence du DBS comme support pour le suivi virologique des patients en milieux décentralisés. Son utilisation a permis de montrer d'autre part des taux d'échecs virologiques élevés indiquant la nécessité de renforcer l'adhérence au traitement. Enfin, nos résultats soulignent l'utilité de prendre davantage en considération les profils de résistance pour initier un traitement de relaisOne of the major barriers to the optimal care of patients undergoing antiretroviral therapy is the limited access to viral load (VL) and genotyping tests, especially in remote areas. These technologies are usually available only at central health facilities in larger cities and plasma is the reference sample. However, plasma or whole blood samples shipment from remote areas to reference lab faces several constraints or even impossible. In order to bring closer patients to reference lab, we have demonstrated the ability of DBS (Dried Blood Spots) collected and shipped in field conditions to provide complete virological monitoring (VL and genotyping). We also documented for the first time, virological outcome of ART and HIV-1 genetic diversity in adult patients followed up in decentralized settings in Senegal, Mali and Guinea Conakry. Overall, despite the low treatment adherence noted sometimes, our findings show no significant differences in the occurrence of virological failure among patients followed up in the central and peripheral health facilities, whatever the country. In Senegal, no integrase inhibitors associated DRM has been found despite the high rate of resistance in patients failing first and second-line treatment. Furthermore, among children born to HIV infected mothers, NNRTI-associated drug resistant mutations (DRM) were more predominant, probably because of systematic use of Nevirapine in MTCT. Our studies also confirm the high genetic diversity of viral subtypes, with the dominance of CRF02_AG in West Africa. This work presented here highlights the feasibility and relevance of DBS as support for the virological monitoring of patients in decentralized settings in West Africa. Furthermore, its use showed high rate of virological failure indicating the need to reinforce adherence to treatment. Finally, our results highlight the utility to considering carefully drug resistance patterns before switching to another ART regimen
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Schilb, Andrew L. "OPTIMIZATION OF NON-VIRAL GENE DELIVERY SYSTEM FOR IMAGE-GUIDED THERAPY FOR TRIPLE NEGATIVE BREAST CANCER." Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1627484657204883.
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Nguyen, Hai Le. "HIV-1 minority variants associated with drug resistance to reverse transcriptase and integrase inhibitors and genetic barrier for the development of resistance to integrase inhibitors." Paris 7, 2012. http://www.theses.fr/2012PA077051.
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Les variants minoritaires résistants (VMR) du VIH-1 aux antirétroviraux n'ont pas été étudiés en Thaïlande. Deux groupes de patients dont le génotypage conventionnel n'a pas montré de mutations associées à la résistance ont été inclus dans l'étude: 104 patients récemment infectés, naïfs de traitement antirétro viral et 22 patients en échec de traitement de première ligne par les inhibiteurs non-nucléosidiques de la transcriptase inverse. Les résultats de pyroséquençage ont montré que la prévalence des variants minoritaires Y181C et M184V dans les 2 groupes est faible en Thaïlande. Le rôle des VMR N155H dans la sélection des profils de résistance au raltégravir (RAL) ont été évalué chez des patients multitraités en échec au RAL. Une PCR allèle spécifique (AS-PCR) a été utilisée pour détecter les mutants N155H. La sélection précoce de cette mutation à des niveaux variés n'influence pas la maintenance de cette mutation pendant l'échec en comparaison avec le passage au double mutant Q148H+Q140S, suggérant que ce mutant N155H n'aurait pas de rôle dans la détermination de profils de résistance. La barrière génétique pour l'évolution de la résistance aux inhibiteurs d'intégrase (INIs) a été comparée entre le sous-type B et CRF01-AE du VIH-1 par l'analyse des 66 mutations associées à la résistance aux INIs dans 144 séquences d'intégrase (109 VIH-1 sous-type CRF01-AE et 35 VIH-1 sous-type B) chez des patients naïfs aux INIs. La plupart des positions d'acides aminés étudiées montrent un haut niveau de conservation, ce qui indique la même barrière génétique entre les sous-types CRF01-AE et BMinority HIV-1 drug resistance bas not been studied in Thailand. Two groups of patients, whose conventional genotyping results showed no drug resistance-associated mutations, were investigated: 104 homosexual men recently infected with HIV-1, naive to antiretroviral treatment and 22 first-line NNRTI-based failures. Pyrosequencing assay was developed to detect and quantify minority Y181C and M184V variants from the patients' plasma samples. 1/104 (0. 96%) and 3/101 (3%) samples were found harboring Y181C and M184V in the group of homosexual men. In patients with first-line treatment failure, one harbored minority Ml84V mutants (4. 5%). Thus, due to such a low prevalence, minority drug résistance test may not be cost-effective for implementing in Thailand. The mechanism of raltegravir (RAL)-resistant evolutions has not been completely elucidated. Because of the emergence of RAL résistance usually initiated with the N155H mutant, we assessed the role of minority N155H-mutated variants in circulating RNA and archived DNA in 5 heavily treated patients experiencing RAL failure and harboring 3 different résistance profiles. No minority N155H-mutated variant was found by allele specific PCR (AS-PCR) in both plasma and whole blood samples collected at baseline and after RAL withdrawal in ail 5 patients. During RAL failure, the mutation N155H was detected at different levels in 3 patients displaying the N155H pathway and gradually declined when the double mutant Q148H+G140S was selected in one patient. In two patients with the Q148H résistance pathway, no N155H variant was identified by AS-PCR in both viral RNA and DNA. The N155H mutants might not play a role in determining different résistance profiles. The genetic barrier, defined by the accumulative number of drug-associated mutations required for the virus to escape drug-selective pressure, is a crucial factor in the development of drug résistance. There are limited data on subtype CRJF01_AE, a predominant isolate in Southeast Asia. The genetic barrier for the evolution of integrase inhibitors (INIs) including RAL, elvitegravir (EVG), and dolutegravir (DTG) résistance was compared between HIV-1 subtypes B and CRF01_AE by analyzing of 66 substitutions associated with INI résistance at 41 amino acid positions in 144 nucleotide sequences (109 HIV-1 subtype CRF01_AE and 35 HIV-1 subtype B) of IN gene derived from INI-naïve patients. Most studied amino acid positions including ail corresponding to RAL and EVG primary mutations show a high degree of conservation, indicating the same genetic barrier between subtypes CRF01_AE and B. Nevertheless, different genetic barriers were observed in two mutations described to be associated with DTG résistance (L101I, A124T) and other five RAL and EVG secondary mutations (V72I, T125K, G140C/S, V201I), which could have an impact on the development of résistance to RAL, EVG, and DTG
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Neto, Jadir Rodrigues Fagundes. "Acompanhamento clínico-laboratorial da utilização de Enfuvirtida em pacientes HIV soropositivos multiexperimentados atendidos nos ambulatórios do Hospital Universitário Pedro Ernesto." Universidade do Estado do Rio de Janeiro, 2012. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=5973.
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A Enfuvirtida(ENF), único inibidor de fusão disponível, representa uma opção interessante aos pacientes com infecção pelo HIV quando utilizada em combinação com outros antirretrovirais, principalmente no tratamento de multiexperimentados com falha virológica e poucas opções terapêuticas. Sua eficácia já comprovada em ensaios clínicos esbarra nas barreiras impostas por sua administração parenteral. Impulsionado por estes dados, avaliamos durante 48 semanas a resposta virológica, a evolução de células T CD4 a possível resistência primária a ENF e o impacto para a adesão do uso subcutâneo da droga em dez pacientes que fazem acompanhamento ambulatorial no Hospital Universitário Pedro Ernesto e que tinham história de mais de dez anos de infecção pelo HIV e uso de ENF no seu esquema terapêutico sugerido por teste de resistência. Todos os pacientes alcançaram ao final do seguimento sucesso terapêutico, mantendo carga viral não detectada, e um incremento médio significativo de linfócitos T CD4. Em relação a uma possível resistência primária, em nenhum dos testes, genotipagem da glicoproteína 41, foi visualizado mutações naturais que pudessem diminuir a ação da ENF. Sobre o manejo do medicamento, preparo e aplicação, observamos que é imprescindível um apoio multidisciplinar para que não haja descontinuação na sua utilizaçãoEnfuvirtide (ENF) is the only fusion inhibitor available. It is an interesting option for patients with HIV infection when used in combination with other antiretroviral drugs, especially in the treatment of multi-experienced patients with virological failure and few therapeutic options. Its effectiveness confirmed in clinical trials finds the barriers in its parenteral administration. Using these data, we evaluated, for 48 weeks, the virological response, evolution of CD4 T cells, the possible primary resistance to ENF and the impact to the subcutaneous use of the drug in ten patients undergoing outpatient monitoring at Hospital Universitário Pedro Ernesto with a history of more than ten years of HIV infection and use of ENF in their therapy, as suggested by resistance testing. All patients have successfully completed the therapy by the end of follow-up with an undetected viral load and a significant average increase of CD4 T lymphocytes. As for a possible primary resistance, neither the genotyping nor the glycoprotein 41 revealed natural mutations that could diminish the effect of ENF. Concerning the management, preparation and application of the drug, we found that a multidisciplinary support is essential to avoid that the drug be discontinued
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Rath, Barbara. "La mégère apprivoisée : élaborer des stratégies pour la gestion de la résistance aux médicaments dans la grippe et l'infection par le virus de l'immunodéficience humaine." Thesis, Besançon, 2012. http://www.theses.fr/2012BESA3012/document.
Full textAbstract:
Le développement de médicaments efficaces contre le virus de l'immunodéficience humaine (VIH) est l'une des plus grandes réussites dans l'histoire médicale récente: lorsque la thérapie combinée est devenu la norme des soins en 1996, une maladie mortelle a été progressivement transformée en une maladie chronique gérable. Les décennies suivantes ont été consacrées à l'élaboration des schémas thérapeutiques consolidés pour les adultes et les enfants, à la prévention de la transmission mère-enfant et à élargir l'accès à la thérapie antirétrovirale dans les pays en développement. La réussite d'un traitement antiviral de l'infection par le VIH est devenue un modèle pour l'élaboration de stratégies de traitement efficaces pour d'autres maladies virales, telles que les hépatites, les infections à herpesviridae, enteroviridae, et la grippe A et B. Cette thèse vise à tracer une ligne continue depuis : (1) de nouveaux modèles in vitro pour simuler un traitement combiné contre un VIH-1 multirésistant afin de promouvoir la sélection du régime le plus/ durable chez les patients en sauvetage thérapeutique, à (2) la meilleure approche e11 termes de coût-bénéfice pour la surveillance de la pharmacorésistance dans les cohortes de patients traités dans des milieux à faibles ressources, et enfin jusqu'à (3) une approche translationnelle vers la gestion du traitement de la grippe et la prédiction du développement- de virus résistants aux médicaments chez les enfants. Elle vise à fournir une synthèse des leçons apprises dans l'optimisation de stratégies de traitements antiviraux et de la prévention des résistances contre le VIH et le virus de fa grippe chez les adultes et les enfantsThe development or efficacious drugs against the human immunodeficiency virus is one of the greatest success stories in the recent medical history: when combination therapy became standard of care after the Vancouver Conference in 1996, a deadly disease was gradually turned into a manageable chronic condition. The following decades have been dedicated to developing consolidated treatment regimens for both adults and children, to the prevention of mother-to-child transmission and to expanding access to antiretroviral therapy (AR1) in developing countries. Subsequently, the success story of antiviral treatment of Hl V infection has become a model for tl1e development of successful treatment strategies for other viral diseases, such as hepatitis and infections with herpesviridae, enteroviridae and influenza A and B. This thesis aims to draw a continuous line from: (1) new in vitro models to simulate comhination therapy against multidrug-resistant HIV-1 promoting the selection of the most sustainable regimen in salvage patients, to (2) a cost-effective approach to monitoring drug resistance in treatment cohorts in low-resource settings, and finally to (3) a translational approach to managing influenza therapy and predicting the development of drug resistant influenza in children. The work presented herein aims to provide a comprehensive view of the lessons learned in optimizing antiviral treatment strategies against HIV and influenza virus in adults and children
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Araya, Seare Tesfamichael. "Support vector machine prediction of HIV-1 drug resistance using The Viral Nucleotide patterns." Thesis, 2007. http://hdl.handle.net/10539/2104.
Full textAbstract:
Student Number : 0213068F -
MSc Dissertation -
School of Computer Science -
Faculty of ScienceDrug resistance of the HI virus due to its fast replication and error-prone mutation is a key factor in the failure to combat the HIV epidemic. For this reason, performing pre-therapy drug resistance testing and administering appropriate drugs or combination of drugs accordingly is very useful. There are two approaches to HIV drug resistance testing: phenotypic (clinical) and genotypic (based on the particular virus’s DNA). Genotyping tests HIV drug resistance by detecting specific mutations known to confer drug resistance. It is cheaper and can be computerised. However, it requires being able to know or learn what mutations confer drug resistance. Previous research using pattern recognition techniques has been promising, but the performance needs to be improved. It is also important for techniques that can quickly learn new rules when faced with new mutations or drugs. A relatively recent addition to these techniques is the Support Vector Machines (SVMs). SVMs have proved very successful in many benchmark applications such as face recognition, text recognition, and have also performed well in many computational biology problems where the number of features targeted is large compared to the number of available samples. This paper explores the use of SVMs in predicting the drug resistance of an HIV strain extracted from a patient based on the genetic sequence of those parts of the viral DNA encoding for the two enzymes, Reverse Transcriptase or Protease, which are critical for the replication of the HIV virus. In particular, it is the aim of this reseach to design the model without incorporating the biological knowledge at hand to enable the resulting classifier accommodate new drugs and mutations. To evaluate the performance of SVMs we used cross validation technique to measure the unbiased estimate on 2045 data points. The accuracy of classification and the area under the receiver operating characteristics curve (AUC) was used as a performance measure. Furthermore, to compare the performance of our SVMs model we also developed other prediction models based on popular classification algorithms, namely neural networks, decision trees and logistic regressions. The results show that SVMs are a highly successful classifier and out-perform other techniques with performance ranging between (94.13%–96.33%) accuracy and (81.26% - 97.49%) AUC. Decision trees were rated second and logistic regression performed the worst.
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Bronze, Michelle Saltao. "In vitro HIV-1 drug resistance phenotyping, genotyping and novel virological failure detection tools for clinical patient management." Thesis, 2014.
Find full textAbstract:
Of the 22.5 million individuals infected with the human immunodeficiency virus (HIV) in
sub-Saharan Africa, 62% of patients requiring treatment had access to highly active
antiretroviral therapy (HAART) in 2011. The delivery of HAART and the appropriate
laboratory monitoring of HIV positive individuals in sub-Saharan African countries has
become a public health priority, an intervention which has and will continue to dramatically
reduce HIV-related morbidity and mortality. Routine laboratory monitoring of HIV infected
individuals should ideally include CD4+ T cell testing to assess when to start ART, viral load monitoring to assess virological failure on ART and when indicated, HIVDR genotyping.However, this is often not implemented in resource limited settings due to challenges such as inadequate infrastructure and laboratory capacity, amongst others. Thus the Affordable Resistance Testing for Africa (ART-A) initiative was established to develop an affordable HIV drug resistance testing (HIVDR) algorithm applicable to Africa. The objective of this study was to evaluate the role of in vitro HIVDR phenotyping in the context of HIV-1 subtype C (the most prevalent circulating subtype in sub-Saharan Africa), genotyping and genotypic interpretation tools using existing algorithms, as well as novel virological failure detection tools for clinical patient management.
Current gold standard HIVDR phenotyping technologies use an HIV-1 subtype B backbone to create recombinant viruses with patient-derived polymerase (protease and partial reverse transcriptase). This backbone could impact on the in vitro phenotyping results of non-B subtypes, and therefore it was deemed necessary to establish the applicability of HIVDR phenotypic testing of subtype C polymerase when a commercially available subtype B backbone is used. One hundred and fourteen HIV-1 subtype C samples were HIVDR phenotyped against 17 antiretroviral drugs using both subtype B and C backbones and showed a high level of concordance between the two backbone phenotypic resistance profiles (95.8%; 1590 of 1660 fold change comparisons). Natural assay variability was largely responsible for discordant results. Results confirmed that HIV-1 phenotypic reverse transcriptase inhibitor drug resistance test interpretation is independent of the virus backbone subtype. No conclusions could be made for protease inhibitor resistance since limited samples from 2nd line failure were available. Subsequently, the HIVDR genotypic and phenotypic results of the 114 patient samples were compared to determine whether genotyping is a viable alternative to phenotyping. Results showed a 92.3% concordance between genotyping and phenotyping of individual drug comparisons for a number of HIVDR profiles. Discrepancies were attributed to phenotypic assay variability in addition to the role of mutation mixtures, which impacted genotypic interpretations. Overall, HIVDR genotyping is a reliable tool to detect and interpret antiretroviral drug resistance in HIV-1 subtype C infected patients, and can thus be used for clinical patient management.
Once the accuracy of HIVDR genotyping was established, the development, validation and evaluation of a potential virological failure assay (ARTA-VFA) and a simplified HIVDR
(ARTA-HIVDRultralight) assay was undertaken. A simplified and conceptually novel approach using a qualitative viral load assay with a pre-determined cut-off that gives a threshold above which virological failure (VF) could be confirmed and below which treatment success was likely, was tested. A real-time PCR (ARTA-VFA) assay was developed which involved the amplification of a short sequence of the HIV-1 LTR region from RNA extracted either from plasma and/or dried blood spots (DBS). The ARTA-VFA was tested on 409 patient samples,and successfully amplified samples from all major HIV-1 group M subtypes with equal specificity. The VF was qualitatively classified as a viral load >1000 RNA copies/ml in plasma samples, and >5000 RNA copies/ml in DBS samples. Comparative testing yielded accurate VF determination for therapy-switching in approximately 93% of clinical cases tested, compared to current gold standard quantitative viral load assays.
A simplified HIVDR genotyping assay (ARTA-HIVDRultralight) targeting the region of RT
harboring all major RT inhibitor resistance mutation positions, thus providing all relevant
susceptibility data for first-line regimen failures was developed and assessed. The ARTAHIVDRultralight assay was designed to be practical, faster, and more affordable, show flexibility with respect to equipment (open platform), use DBS or plasma as starting material and amplify and sequence a smaller amplicon (RT). The assay performed well when compared to the in-house assay used in the laboratory at the time for both 212 plasma and 25 DBS samples, yielding identical mutations and subsequent resistant profiles. Furthermore, a theoretical in silico exercise to investigate the consequences of using 125,329 shortened RT genotype (ARTA-HIVDRultralight) as compared to full-length RT sequences showed >95% and >90% concordance when using the Stanford HIVdb algorithm and the virco®TYPE tool, respectively. Differences noted were minor and unlikely to have any impact on clinical decision-making. Overall, this study illustrated that the short RT sequences can be reliably used to generate HIVDR genotypes using the Stanford HIVdb and virco®TYPE algorithms and reduce sequencing costs substantially. A field evaluation using the ARTA-VFA and ARTA-HIVDRultralight on 288 clinical samples was conducted, showing that the accuracy and precision of both assays (using 248 plasma or 40 DBS sampling methods) compared well to the reference methodology, thereby extending access of testing to more remote settings.These assays were designed to either be used as a testing strategy of initially assessing VF,and once confirmed performing an HIVDR assay, or alternatively to be used separately as
stand-alone, or within different laboratory tiers in resource limited settings. It is envisaged
that the ARTA-VFA could be used in the middle laboratory tier, and if confirmatory, patient
samples can be referred to a reference laboratory with the available infrastructure for HIVDR testing using the ARTA-HIVDRultralight. Lastly, an automated sequence analysis and editing software for use in correct base calling of nucleotide/mutation mixtures in HIVDR genotyping was validated on 1624 sequences. Compared to reference software, where interpretation is often operator dependent, this software performed extremely well, with minor discrepancies noted. The automated software can be used to reduce subjectivity, time taken for analysis which is often the rate-limiting step and thus improving the turn-around time and clinical relevance of HIVDR genotyping.
Overall, the results obtained describe the validation of using HIVDR genotyping as an
alternative tool to phenotyping, and the subsequent development and validation of simple,
affordable, "open-platform" alternatives to currently used methods for virological failure
monitoring, and accommodate a centralized approach to HIVDR with DBS testing in
resource limited settings.
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Giandhari, Jennifer. "The role of the protease cleavage sites in viral fitness and drug resistance in HIV-1 subtype C." Thesis, 2010. http://hdl.handle.net/10413/9956.
Full textAbstract:
There is an increasing number of patients failing second line highly active antiretroviral therapy
(AZT, DDI and LPV/r) in South Africa, where HIV-1 subtype C predominates. Mutations at gag
cleavage sites (CS) have been found to correlate with resistance mutations in protease (PR).
Therefore, it is important to collect data on subtype C protease and gag sequences from patients
as these mutations may affect the efficacy of protease inhibitor (PI) containing drug regimens.
In this study, 30 subtype-C infected second-line failures were genotyped using the ViroSeqTM
resistance genotyping kit and the gag region from these isolates were then characterised. These
sequences were then compared to 30 HIV-1 subtype C infected first-line failures (PI-naïve) and
subtype B, C and group M naïve sequences that were downloaded from the Los Alamos
Sequence Database. Amino acid diversity at the CS was measured using Mega version 4.0. To
investigate the effect of CS mutations on replication capacity, a mutation was introduced by
site-directed mutagenesis (Stratagene’s QuikChange Site-Directed Mutagenesis kit).
Of the 30 second-line failures that we genotyped, only 16 had resistance mutations in PR and 23
in gag. The most frequent major PI mutations were: I54V/L, M46I, V82A, and I84V and in gag
CS were V390L/I and A431V. Interestingly the A431V mutation significantly correlated with
protease mutations M46I/L, I54V and V82A. The virus carrying the A431V mutation in vitro
was found to have a lower replication capacity compared to the wild type.
These findings emphasize the need for further investigation of gag mutations and their
contribution to the evolution of HIV resistance to PIs.Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2010.
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Gammon, Donald Brad. "Vaccinia virus DNA polymerase and ribonucleotide reductase their role in replication, recombination and drug resistance /." 2010. http://hdl.handle.net/10048/860.
Full textAbstract:
Thesis (Ph.D.)--University of Alberta, 2010.A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology, Medical Microbiology and Immunology. Title from pdf file main screen (viewed on January 10, 2010). Includes bibliographical references.
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Gordon, Michelle Lucille. "Molecular characterization of HIV-1 Subtype C strains from KwaZulu-Natal, South Africa, with a special emphasis on viral fitness and drug resistance." Thesis, 2004. http://hdl.handle.net/10413/2533.
Full textAbstract:
As South Africa begins its National HIV-1 treatment program, it is urgent that we collect data that will help define the phylogenetic relationships, transmissibility and drug responsiveness of C viruses. In this thesis, data is presented on the genetic diversity of locally circulating drug naive subtype C strains, as an indication of their natural susceptibility to antiretroviral drugs, prior to the national roll-out of antiretroviral therapy. At the time this thesis was initiated, antiretroviral therapy was only available in South Africa in a few clinical trials and in the private sector, and it was therefore difficult to obtain large numbers of samples from treatment-experienced patients. Nevertheless, valuable information on the prevalence and patterns of resistance mutations in subtype C infected patients was obtained from small studies on patients receiving HAART, concomitant HAART and TB treatment, HAART and treatment for Kaposi Sarcoma, and single dose nevirapine for the prevention of mother-to-child transmission of HIV-1 infection. The results show that the general antiretroviral drug naive population do not harbour any major resistance-associated mutations to the currently available protease and reverse transcriptase inhibitors, with no differences in genetic variation between the different ethnic groups infected with subtype C. Phenotyping of some of these isolates showed that they were susceptible to the available protease and reverse transcriptase inhibitors, and hyper-susceptible to the protease inhibitor, Lopinavir. Phylogenetic analysis of recent and retrospective subtype C isolates showed that there are multiple lineages of subtype C viruses circulating in South Africa, indicative of multiple introductions of subtype C across its many borders. Polymorphisms in the protease, reverse transcriptase and C2-V5 region of envelope in these drug naive samples lead to significant variation in the number, type and location of potential phosphorylation sites. There was also variation in the cleavage sites controlling the initiation and rate of Gag and Gag-Pol processing (p2/NC) and the activation of protease (TFP/p6gag) suggesting that there may be important differences in the way that B and C viruses regulate polyprocessing and virion assembly. Similar to studies on subtype B, 10 to 18% of the patients on HAART developed drug resistance. However, those on concomitant HAART and TB treatment developed resistance as early as one month after starting treatment. Generally, the resistance mutations that were seen were consistent with those seen in treatment experienced subtype B isolates. Of note was the high level of resistance to the entire class of NNRTIs. This could be reflective of the predominant use of NNRTI-based regimens, as well as the low genetic barrier in this class of drugs. The NNRTI mutations included the V106M mutation that is considered a signature mutation of EFV experienced subtype C isolates. Resistance was high (40%) in mothers and infants 6 weeks after each received a single dose of NVP. K103N was most common mutation in the mothers, while Y181C was most common in the infants. Of note were the changes in functional properties caused by these mutations, by the introduction or alteration of putative myristoylation and phosphorylation sites in the RT. Taken together, these data suggests that the pattern of resistance in African patients will be similar to that observed for the treatment of subtype B infection. However, patients should be closely monitored for viral rebound very early on in treatment. Also, given the high rate of resistance in mothers and infants after single dose NVP, the search for safer regimens to prevent MTCT should be intensified. Although the mechanisms are unknown, our results indicate that several of the phosphorylation-related substitutions in the pol and env genes of KZN and other C viruses are highly conserved and positively selected. It will be important to determine whether these sites play an important role in the replicative capacity and proteolytic processing of C viruses, and in viral entry. These data provide important benefits for public health policy and planning and for future patient treatment management.Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2004.
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Phathagi, Muendi Tshililelwa. "Construction of an HIV-1 subtype C ventor system for phenotypic drug resistance studies." Diss., 2015. http://hdl.handle.net/11602/301.
Full text