28 research outputs found

    Highly Accurate Structure-Based Prediction of HIV-1 Coreceptor Usage Suggests Intermolecular Interactions Driving Tropism

    Get PDF
    HIV-1 entry into host cells is mediated by interactions between the V3-loop of viral glycoprotein gp120 and chemokine receptor CCR5 or CXCR4, collectively known as HIV-1 coreceptors. Accurate genotypic prediction of coreceptor usage is of significant clinical interest and determination of the factors driving tropism has been the focus of extensive study. We have developed a method based on nonlinear support vector machines to elucidate the interacting residue pairs driving coreceptor usage and provide highly accurate coreceptor usage predictions. Our models utilize centroid-centroid interaction energies from computationally derived structures of the V3-loop:coreceptor complexes as primary features, while additional features based on established rules regarding V3-loop sequences are also investigated. We tested our method on 2455 V3-loop sequences of various lengths and subtypes, and produce a median area under the receiver operator curve of 0.977 based on 500 runs of 10-fold cross validation. Our study is the first to elucidate a small set of specific interacting residue pairs between the V3-loop and coreceptors capable of predicting coreceptor usage with high accuracy across major HIV-1 subtypes. The developed method has been implemented as a web tool named CRUSH, CoReceptor USage prediction for HIV-1, which is available at http://ares.tamu.edu/CRUSH/

    A genotypic method for determining HIV-2 coreceptor usage enables epidemiological studies and clinical decision support

    Get PDF
    Background: CCR5-coreceptor antagonists can be used for treating HIV-2 infected individuals. Before initiating treatment with coreceptor antagonists, viral coreceptor usage should be determined to ensure that the virus can use only the CCR5 coreceptor (R5) and cannot evade the drug by using the CXCR4 coreceptor (X4-capable). However, until now, no online tool for the genotypic identification of HIV-2 coreceptor usage had been available. Furthermore, there is a lack of knowledge on the determinants of HIV-2 coreceptor usage. Therefore, we developed a data-driven web service for the prediction of HIV-2 coreceptor usage from the V3 loop of the HIV-2 glycoprotein and used the tool to identify novel discriminatory features of X4-capable variants. Results: Using 10 runs of tenfold cross validation, we selected a linear support vector machine (SVM) as the model for geno2pheno[coreceptor-hiv2], because it outperformed the other SVMs with an area under the ROC curve (AUC) of 0.95. We found that SVMs were highly accurate in identifying HIV-2 coreceptor usage, attaining sensitivities of 73.5% and specificities of 96% during tenfold nested cross validation. The predictive performance of SVMs was not significantly different (p value 0.37) from an existing rules-based approach. Moreover, geno2pheno[coreceptor-hiv2] achieved a predictive accuracy of 100% and outperformed the existing approach on an independent data set containing nine new isolates with corresponding phenotypic measurements of coreceptor usage. geno2pheno[coreceptor-hiv2] could not only reproduce the established markers of CXCR4-usage, but also revealed novel markers: the substitutions 27K, 15G, and 8S were significantly predictive of CXCR4 usage. Furthermore, SVMs trained on the amino-acid sequences of the V1 and V2 loops were also quite accurate in predicting coreceptor usage (AUCs of 0.84 and 0.65, respectively). Conclusions: In this study, we developed geno2pheno[coreceptor-hiv2], the first online tool for the prediction of HIV-2 coreceptor usage from the V3 loop. Using our method, we identified novel amino-acid markers of X4-capable variants in the V3 loop and found that HIV-2 coreceptor usage is also influenced by the V1/V2 region. The tool can aid clinicians in deciding whether coreceptor antagonists such as maraviroc are a treatment option and enables epidemiological studies investigating HIV-2 coreceptor usage. geno2pheno[coreceptor-hiv2] is freely available at http://coreceptor-hiv2.geno2pheno.org

    Computational approaches for improving treatment and prevention of viral infections

    Get PDF
    The treatment of infections with HIV or HCV is challenging. Thus, novel drugs and new computational approaches that support the selection of therapies are required. This work presents methods that support therapy selection as well as methods that advance novel antiviral treatments. geno2pheno[ngs-freq] identifies drug resistance from HIV-1 or HCV samples that were subjected to next-generation sequencing by interpreting their sequences either via support vector machines or a rules-based approach. geno2pheno[coreceptor-hiv2] determines the coreceptor that is used for viral cell entry by analyzing a segment of the HIV-2 surface protein with a support vector machine. openPrimeR is capable of finding optimal combinations of primers for multiplex polymerase chain reaction by solving a set cover problem and accessing a new logistic regression model for determining amplification events arising from polymerase chain reaction. geno2pheno[ngs-freq] and geno2pheno[coreceptor-hiv2] enable the personalization of antiviral treatments and support clinical decision making. The application of openPrimeR on human immunoglobulin sequences has resulted in novel primer sets that improve the isolation of broadly neutralizing antibodies against HIV-1. The methods that were developed in this work thus constitute important contributions towards improving the prevention and treatment of viral infectious diseases.Die Behandlung von HIV- oder HCV-Infektionen ist herausfordernd. Daher werden neue Wirkstoffe, sowie neue computerbasierte Verfahren benötigt, welche die Therapie verbessern. In dieser Arbeit wurden Methoden zur Unterstützung der Therapieauswahl entwickelt, aber auch solche, welche neuartige Therapien vorantreiben. geno2pheno[ngs-freq] bestimmt, ob Resistenzen gegen Medikamente vorliegen, indem es Hochdurchsatzsequenzierungsdaten von HIV-1 oder HCV Proben mittels Support Vector Machines oder einem regelbasierten Ansatz interpretiert. geno2pheno[coreceptor-hiv2] bestimmt den HIV-2 Korezeptorgebrauch dadurch, dass es einen Abschnitt des viralen Oberflächenproteins mit einer Support Vector Machine analysiert. openPrimeR kann optimale Kombinationen von Primern für die Multiplex-Polymerasekettenreaktion finden, indem es ein Mengenüberdeckungsproblem löst und auf ein neues logistisches Regressionsmodell für die Vorhersage von Amplifizierungsereignissen zurückgreift. geno2pheno[ngs-freq] und geno2pheno[coreceptor-hiv2] ermöglichen die Personalisierung antiviraler Therapien und unterstützen die klinische Entscheidungsfindung. Durch den Einsatz von openPrimeR auf humanen Immunoglobulinsequenzen konnten Primersätze generiert werden, welche die Isolierung von breit neutralisierenden Antikörpern gegen HIV-1 verbessern. Die in dieser Arbeit entwickelten Methoden leisten somit einen wichtigen Beitrag zur Verbesserung der Prävention und Therapie viraler Infektionskrankheiten

    Human Immunodeficiency Virus Type 1 Envelope Protein Interaction with Host Cell Coreceptor C-X-C Chemokine Receptor Type 4

    Get PDF
    Human Immunodeficiency Virus Type 1 (HIV-1) envelope protein is the sole determinant for viral entry and tropism. The ability of HIV-1 to infect susceptible host cells depends on the ability of its envelope protein to engage host cell receptor CD4 and coreceptor C-C Chemokine Receptor Type 5 (CCR5) and/or C-X-C Chemokine Receptor Type 4 (CXCR4). Most naturally occurring infections start with a single CCR5-tropic virion. In approximately 50% of HIV-1 Clade B infected patients, the viral population spontaneous develops the ability to utilize CXCR4 at a late disease stage, and this coreceptor shift corresponds to a poor prognosis for the patients. Clinical application of a CCR5 antagonist drug accelerates this coreceptor shift process. Despite the important implications of HIV-1 tropism on disease pathogenesis, prognosis, and treatment, molecular mechanisms for coreceptor shift and the contributing envelope determinants have not yet been clearly defined due to the dynamic, multimeric, multi-step nature of envelope-coreceptor interaction. In order to better understand the regions on envelope that are important for coreceptor specificity, we studied a panel of HIV-1 envelope amplicon samples from patients who experienced coreceptor shift and treatment failure in a CCR5 antagonist clinical trial. In order to further understand the structure-function relationship of HIV-1 envelope, we utilized both in-depth single clone analysis to fully characterize the functional attributes of individual envelope isolates, and high-throughput deep sequencing analysis to investigate the global envelope variant landscape before and after treatment. In the single clone analysis, we applied both tissue-culture based phenotypic tropism characterization, as well as sequence-based genotypic analysis towards 97 single envelope clones isolated from four samples of two study subjects. Unique quasispecies composition were found post coreceptor shift in two individuals who had different courses of disease. In addition, one subject showed drastic sequence variation between isolates from before and after treatment, and a highly homogeneous viral population post treatment. This suggested a rapid shift to CXCR4-using variants that accounted for failure to respond to CCR5 antagonist treatment. In the deep sequencing analysis, we developed a novel comprehensive approach to sequence a large number of phenotypic validated variants on two next-generation sequencing platforms. This approach offered an unprecedented view of the viral quasispecies landscape in vivo, as well as on the dynamic population change in response to drug selection. Regions of interest that strongly associated with the usage of CXCR4 coreceptor were identified, including previously reported coreceptor specificity sites within and outside V3, the CD4 binding site, and gp41-gp120 interaction site within the same protomer and between neighboring protomers in the envelope trimer spike. These findings have potentially implications in rational design of better coreceptor inhibitors, and development of more accurate predictive algorithms for HIV-1 envelope tropism

    Characterization of HIV-2 susceptibility to protease and entry inhibitors and identification of envelope determinants of coreceptor usage, cell tropism and antibody neutralization

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

    Mécanismes de résistance aux inhibiteurs de CCR5

    Get PDF
    Le travail décrit dans ce manuscrit vise à caractériser les voies de résistance aux inhibiteurs de CCR5. Lors d’une première étape, nous avons développé un test phénotypique clonal nous permettant d’une part d’identifier le tropisme viral et d’autre part de mesurer la résistance aux inhibiteurs des CCR5. Des virus à tropisme R5 ou X4 représentant aussi peu que 0,4% d’un mélange de populations virales sont détectables par ce test, démontrant ainsi sa sensibilité. De plus, grâce à son approche clonale, cette technique permet de différencier les virus à tropisme double de populations virales mixtes. Par la suite, nous avons étudié l’impact des mutations dans les régions variables de la protéine gp120 de l’enveloppe du virus VIH-1 sur la résistance aux inhibiteurs de CCR5. Pour ce faire, nous avons généré des virus résistants par passage des isolats CC1/85 et BAL, en présence de concentrations sous-inhibitrices de maraviroc (MVC) et vicriviroc (VCV). Après quelques passages du virus CC1/85 en présence de MVC, certaines sont apparues dans differentes régions de la gp120. Par la suite, nous avons sélectionné trois mutations dans les domaines variables de la gp 120, V169M en V2, L317W en V3 et I408T en V4 pour construire des virus contenant des mutations simples, doubles et triples afin d’évaluer la contribution des mutations individuelles ou combinées au phénotype de résistance. Nous avons déterminé la sensibilité de chaque mutant à MVC et VCV, le pourcentage d’infectivité et le tropisme viral par rapport au phénotype sauvage. Tous les mutants ont conservé le tropisme R5 et ont montré une diminution d’infectivité par rapport au contrôle. Nos résultats ont montré que les mutants qui portent des mutations en V4 (I408T) ont eu le plus d'impact sur la susceptibilité au MVC. Finalement, nous avons voulu évaluer l’activité antivirale d’un nouvel inhibiteur de CCR5, VCH-286 avec d’autres inhibiteurs de CCR5 tels que MVC et VVC ainsi que ses interactions avec des médicaments représentatifs de différentes classes d’antirétroviraux ARV employés en clinique pour traiter le HIV/SIDA., afin d’évaluer si ces médicaments pourraient être utilisés dans un même régime thérapeutique. Nous avons tout d’abord évalué indépendamment l’activité antivirale des trois inhibiteurs de CCR5 : VCH-286, MVC et VVC. Par la suite nous avons évalué les interactions de VCH-286 avec MVC et VVC. Finalement nous avons évalué les interactions de VCH-286 avec d’autres médicaments antirétroviraux. Ces études ont montré que VCH-286 est un inhibiteur puissant de CCR5 avec une activité antivirale in vitro de l’ordre du nanomolaire et des interactions médicamenteuses favorables avec la majorité des ARV tels que les inhibiteurs de transcriptase inverse, de protéase, d’intégrase, et de fusion employés en clinique pour traiter le VIH/SIDA et des interactions allant de synergie à l'antagonisme avec les inhibiteurs de CCR5. Nos résultats montrent que la plasticité de l’enveloppe virale du VIH-1 a des répercussions sur la résistance aux inhibiteurs de CCR5, le tropisme et la possible utilisation de ces molécules en combinaison avec d’autres molécules appartenant à la même classe.The work described in this manuscript aimed to characterize the resistance pathways to CCR5 inhibitors. We first developed a phenotypic assay to identify viral tropism and to measure the resistance to CCR5 inhibitors. This assay detects R5 tropic viruses or X4 when they represent as little as 0.4% in a mixture of viral populations, demonstrating its robustness and sensitivity. Based on its clonal approach, this assay can differentiate the truly dual-tropic viruses from mixed viral populations. We then studied the impact of mutations in the variable regions of gp120 envelope protein of HIV-1 virus on resistance to CCR5 inhibitors. To do this, resistant viruses were generated by passage of CC1/85 and BAL isolates in the presence of sub-inhibitory concentrations of maraviroc (MVC) and vicriviroc (VCV). Following some passages of the CC1/85 virus in the presence of MVC, some mutations were identified in differents regions of the gp120. We further selected three mutations in the variable domains of gp120, V169M in V2, L317W in V3 and I408T in V4 to construct viruses containing single, double and triple mutations to assess the contribution of individual or combined mutations in the resistance phenotype to MVC and VCV. We determined the sensitivity of each mutant to MVC and VCV, the tropism and the percentage of infectivity compared to wild type. Our results showed that the sequences that carry mutations in the V4 domain I408T, had the most impact on susceptibility to MVC. Finally, we aimed to evaluate the antiviral activity of a new CCR5 inhibitor, VCH-286 and its interaction with representative drugs from different classes of antiretroviral (ARVs) such as reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors and fusion inhibitors used in clinic to treat HIV/AIDS and other CCR5 inhibitors such as MVC and VVC to assess whether these drugs could be used together within the same treatment regimen. To answer this question, we first evaluated the antiviral activity of the three CCR5 inhibitors: VCH-286, MVC, and VVC. We then evaluated the interactions of VCH -286 with MVC VVC. We finally evaluated the interactions of VCH -286 with other ARV drugs These studies showed that VCH-286 is a potent inhibitor of R5 viruses with antiviral activity at the nanomolar range and favorable drug interactions with the majority of ARVs such as reverse transcriptase, protease, integrase and fusion inhibitors employed clinically to treat HIV/AIDS. The combinations of CCR5 inhibitors have interactions ranging from synergy to antagonism. Our results show that the plasticity of the viral envelope of HIV-1 affects resistance to CCR5 inhibitors, its tropism and the potential combination of these drugs

    Classificadores de regressão logística, Naive Bayes e Random Forest na análise do Tropismo do HIV-1 de subtipo B

    Get PDF
    The development of coreceptor antagonists – such as maraviroc – for HIV treatment has made mandatory the clinical determination of viral coreceptor usage prior to rescue therapy. Technical issues presented by TrofileTM, the gold standard phenotypic assay, hindered its use as a routine diagnostic tool. This fact has lead to the development of genotypic algorithms, whose evaluations are based on DNA sequences of the V3 region of HIV-1 gp120. These algorithms proved to be cheaper, easier to use, and less time consuming than the phenotypic method. One of them, geno2pheno has also gained widespread use since it showed 86.5% predictive concordance with TrofileTM. The present project aimed to develop accurate classification models based on V3 sequence information. For this, 2,109 DNA sequences of V3 region from HIV-1 subtype B were used. Data labeled with geno2pheno’s results were then modeled by methods such as logistic regression, naive Bayes and random forest. All classifiers presented good predictive outputs, however random forest models showed the best discriminative performance, in the form of significant AUC results. These outcomes encourage us to continue the development of an easy to use and accurate algorithm for HIV-1 tropism diagnosis, capable of guiding clinical decision making regarding the use of coreceptor antagonists in HIV-1 treatment.O desenvolvimento de antagonistas de correceptores – como o maraviroque – para o tratamento anti-HIV tornou mandatória a determinação clínica do tropismo viral previamente às terapias de resgate. Aspectos técnicos do TrofileTM, o ensaio fenotípico referencial, dificultaram o seu uso como ferramenta de rotina para este diagnóstico. Isto levou ao desenvolvimento de algoritmos genotípicos, cujas avaliações são baseadas em sequências genéticas da região V3 da gp120 do HIV-1. Tais algoritmos se mostraram opções menos dispensiosas de custo e tempo, além de serem mais práticos para o uso na rotina clínica do que o ensaio fenotípico. Dentre eles, o geno2pheno começou a ser amplamente utilizado após apresentar uma concordância preditiva de 86,5% com o TrofileTM. O presente projeto visou desenvolver modelos classificadores acurados, baseados em informações de sequências V3. Para isto, foram utilizadas 2.109 sequências de DNA da região V3 do HIV-1 de subtipo B. As sequências com os resultados do geno2pheno foram então modeladas pelos métodos de regressão logística, naive Bayes e random forest. Todos os classificadores apresentaram bons resultados preditivos, porém os modelos de random forest obtiveram o melhor desempenho discriminativo, sob a forma de resultados significativos de AUC. Tais resultados são encorajadores para a continuação do desenvolvimento de um algoritmo acurado e de uso prático para a predição clínica do tropismo viral, capaz de orientar a tomada de decisão em relação à utilização de antagonistas de correceptores no tratamento do HIV-1

    HIV-1 Drug Resistance to Integrase Strand Transfer Inhibitors in HIV-1 non-B Subtypes

    Get PDF
    Human immunodeficiency syndrome (HIV-1) has infected over 75 million people and over 35 million have succumbed to virus related illnesses. Despite access to a variety of antiretroviral therapy (ART) options, ART programs have been disproportionally spread in the world with low-and middle-income countries (LMICs) facing challenges to access the most potent ART options. With less potent ART remaining in use in LMICs, HIV-1 drug resistance (HIVDR) presents a growing challenge in LMICs. Since approval of the first-generation integrase strand transfer inhibitor (INSTIs), Raltegravir (RAL) in 2007, INSTIs remain the best choice as a backbone of ART. Access to second generation INSTIs, Dolutegravir (DTG) and bictegravir (BIC) in LMICs is based on need and not on a full evaluation of the effectiveness of these treatments in patients infected with non-B HIV-1 subtypes. To address this challenge of limited INSTIs associated HIVDR data in non-B HIV subtypes, we first screened for the presence of INSTIs associated drug resistance mutations (DRMs) in ART naïve and experienced patients in Uganda using Sanger and Illumina sequencing. In Uganda, 47% of patients failing on RAL carry resistance to RAL-and elvitegravir (EVG), and only 4% harbor resistant virus to DTG. A panel of recombinant viruses from patient-derived HIV-1 integrases carrying resistant mutations was created and tested for susceptibility to a panel of INSTIs: EVG, RAL, DTG, BIC, and CAB. The virus carrying N155H or Y143R/S was susceptible to DTG, BIC, and CAB but highly resistant to RAL and EVG (\u3e50-fold change). Two patients, one with E138A/G140A/Q148R/G163R and one with E138K/G140A/S147G/Q148K, displayed the highest reported resistance to RAL, EVG (FC, \u3e1000) and even DTG (FC, \u3e100), BIC (FC, 60-\u3e100), and CAB (FC, 429-\u3e1000). All viruses had impaired replication fitness and \u3c50% reduction in integration capacity. We further determined potential novel polymorphisms associated with INSTI resistance in HIV-1 subtype A and D using simple vector machine analysis. The identified I208L and I203M, did not show reduced susceptibility to RAL or DTG with 1.3-1.8-fold and 1-1.4-fold observed, respectively. Further investigation is required to determine how these novel mutations influence susceptibility to INSTIs in HIV-1 subtype A and D infected patients

    Ein Toolkit zur Quantifizierung der Stichprobenqualität von Molekül-Dynamik Trajektorien: eine Studie über hochflexible Biomoleküle

    Get PDF
    In this computational work, we investigate the sampling of molecular dynamics (MD) simulations of the two highly flexible biomolecules: Methionine-enkephalin (Met-Enkephalin) and the third variable loop (V3) of the glycoprotein 120 (gp120) from the human immunodeficiency virus type-1 (HIV-1). The conformational dynamics of the three-dimensional (3D) protein structures are of central importance for the biomolecular function. A common possibility to obtain these dynamics at atomic resolution are MD simulations. But reaching a converged MD sampling in adequate time is limited by the huge conformational space of flexible systems. Moreover, an automatic sampling validation is still not established as settled protocol in today's MD studies. Furthermore, existing tools aim primarily to investigate single trajectory convergence which is not always practical for flexible molecules. But in fact, a universal assessment is necessary to classify, whether the sampling is sufficient or not. Otherwise the extracted thermodynamic results are completely meaningless. The aim of this work is to develop a toolkit to quantitatively assess the MD sampling quality for flexible systems. This toolkit is freely available at https://github.com/MikeN12/PySamplingQuality. We use diverse sets of trajectories with different initial conformations along with enhanced sampling techniques such as accelerated MD (aMD) and scaled MD (sMD). These distort the energy landscape to ease conformational transitions. The sampling is assessed by two new quantities, the conformational Oconf and density overlap Odens, including also the cluster number Nc and cluster distribution entropy Sc. These new overlap quantities measure the self-consistency of sampling as a necessary condition for complete sampling. We use Met-Enkephalin as benchmarking system because of its small size but non-trivial dynamics. Our tool reveals that the MD sampling of already such a small molecule converges in a microsecond regime. Furthermore, we can show that aMD is the most efficient algorithm to assess the convergence and also to detect wrong sampling. However, Odens analysis comparing MD with aMD/sMD reveals that we have not completely corrected the bias from enhanced sampling. Therefore, Odens can also be used to compare different methods. On the other hand, V3 demonstrates that much more resources must be spent to achieve convergence compared to those generally invested today. The results highlight the necessity of a multi-trajectory approach to detect incomplete sampling. Altogether, we are able to generate a universally and easily applicable toolkit to assess the MD sampling quality of any kinds of multi-trajectory experiments using certain error estimates and decide, whether the extracted thermodynamic properties are correct or not.In dieser Arbeit wird das Sampling von Molekulardynamik (MD) Simulationen von zwei flexiblen Biomolekülen untersucht: Methionin-Enkephalin (Met-Enkephalin) und dem dritten variablen Loop (V3) des Glykoproteins 120 (gp120) des Humanen Immundefizienz-Virus Typ-1 (HIV-1). Die Dynamik von drei-dimensionalen (3D) Protein-Strukturen ist von zentraler Bedeutung für die Beschreibung der biomolekularen Funktion. Die Dynamik wird mittels MD Simulationen auf atomarem Level untersucht. Das Erreichen eines konvergierten MD Samplings in adäquater Zeit ist jedoch durch den riesigen Konformationsraum von flexiblen Molekülen begrenzt. Des Weiteren ist eine automatische Validierung des Samplings bisher nicht etabliert in heutigen MD Studien, und existierende Verfahren konzentrieren sich vorwiegend auf die Konvergenzanalyse einzelner Trajektorien. Dies ist für flexible Moleküle problematisch. Dabei ist es notwendig ein ausreichendes Sampling zu quantifizieren, ansonsten sind berechnete thermodynamische Größen bedeutungslos. Das Ziel dieser Arbeit ist die Entwicklung eines Toolkits, welches die Samplingqualität von MD Simulationen von flexiblen Systemen quantifiziert. Dieses ist frei verfügbar unter https://github.com/MikeN12/PySamplingQuality. Hierzu werden verschiedene Sätze von Trajektorien aus verschiedenen Startkonformationen und sogenannte Enhanced Sampling Algorithmen wie accelerated MD (aMD) und scaled MD (sMD) kombiniert. Diese modifizieren die Energielandschaften um Übergänge zu vereinfachen. Die Samplingqualität wird durch zwei neue Messungen quantifiziert, dem Konformations- Oconf und Dichteüberlapp Odens, unter Hinzunahme der Clusteranzahl Nc und der Entropie der Clusterverteilung Sc. Diese neuen Überlappgrößen klassifizieren die Selbstkonsistenz. Met-Enkephalin wird als Testsystem verwendet, aufgrund dessen geringer Peptidlänge aber dennoch hochflexiblen Verhaltens. Unser Tool zeigt, dass bereits ein so kleines Molekül Simulationen von Mikrosekunden zur Konvergenz des Samplings benötigt. Weiterhin gilt, dass aMD sowohl Konvergenz als auch ungenügendes Sampling am schnellsten erkennt. Dennoch hat der Vergleich von Odens zwischen MD und aMD/sMD gezeigt, dass die Modifikation des Enhanced Samplings nicht vollständig wiederhergestellt werden konnte. Dies kann jedoch mittels Odens untersucht werden. V3 hingegen beweist, dass viel mehr Ressourcen als gewöhnlich notwendig sind, um Konvergenz zu erhalten. Die Ergebnisse unterstreichen die Notwendigkeit eines Multitrajektorien Ansatzes, um ungenügendes Sampling eindeutig zu erkennen. Zusammenfassend ist es mit dem Toolkit möglich, das Sampling von Multitrajektorie-Experimenten zu validieren, unter der Angabe von Fehlerabschätzungen, und zu entscheiden, ob die berechneten thermodynamischen Größen korrekt beschrieben werden

    Targeting therapeutic T cells to the bone marrow niche

    Get PDF
    Anti-cancer immunotherapies aim to mediate a specific response targeting malignant cells without accompanying damage to normal tissue associated with conventional therapies, but induction of T cell differentiation and exhaustion enables successful tumour progression. In this thesis I will explore different means of enhancing the accumulation and function of therapeutic CD8 T cells, as a means of achieving functional cure through persisting immunological memory. I will show that the key features of T cell memory can be imprinted upon CD8+ T cells by enhancing homing to specific organs, enabling privileged access to cell-mediated factors. The interaction between the chemokine receptor CXCR4 and the ligand CXCL12/SDF-1 is required for successful homing of haematopoietic stem cells (HSCs) to stromal niches within the bone marrow (BM). The bone marrow is known to be a unique organ for immunological memory, including memory T cells. I hypothesised that replicating this bone marrow homing interaction in CD8+ T cells would preferentially generate memory T cells. I demonstrate through in vivo imaging and flow cytometric analyses that T cells over-expressing CXCR4 accumulate preferentially in the BM near vascular-associated CXCL12+ cells, retain a less differentiated central memory phenotype despite repeated antigenic stimulation, and produce enhanced effector cytokines on restimulation. Compared to control T cells, these cells demonstrate lower expression of exhaustion and senescence markers, suggesting the capacity for long-term persistence after activation. I go on to show that numerical accumulation and many of these functional attributes are dependent upon cell-extrinsic expression of IL-15Rα. TCXCR4 demonstrate heightened graft-versus-tumour effects in allogeneic bone marrow transplant models of B-cell lymphoma in comparison to control T cells. I provide evidence that this anti-tumour effect is mediated by enhanced functional capacity rather than numerical accumulation or out-competing immunosuppressive populations. In summary, this strategy offers a tractable means of enhancing T cell engraftment, persistence and function, with potential for cross-platform therapeutic applications including anti-cancer immunotherapy
    corecore