Modellierung von Verteilungskoeffizienten pharmazeutischer Wirkstoffe in kolloidalen Systemen

Die vorliegende Arbeit beschäftigt sich mit der Modellierung von Verteilungskoeffizienten verschiedener Substanzen, insbesondere von pharmazeutischen Wirkstoffen, in biologisch relevanten Systemen. Das Ziel ist es, ein Modellierungsvorgehen zu entwickeln, welches die Vorhersage der Verteilung organischer Substanzen zwischen einer unterschiedlich komplexen hydrophoben (Oktanol, Mizellkern, Membran) und einer hydrophilen Phase erlaubt. Grundlage der Modellierung ist das COSMO-RS Modell, welches die a-priori Vorhersage der thermodynamischen Eigenschaften aller beteiligten Komponenten basierend auf der Molekülstuktur und damit die Berücksichtigung von Konformationen eines Moleküls ermöglicht. Zuerst wurde die Modellierung anhand des einfachen und gut untersuchten Systems Oktanol/Wasser erarbeitet und validiert. Dabei wurde der Einfluss von Konformeren der Lösungsmittel und des gelösten Stoffes (Solute) untersucht. Es wurde gezeigt, das einzelne Konformere der Lösungsmittel- und Solutemoleküle einen starken Einfluss auf die Qualität der vorhergesagten Verteilungskoeffizienten besitzen. Eine zuverlässige a-priori Vorhersage von Verteilungskoeffizienten ist möglich, wenn bei der Modellierung gewichtete Mischungen der Lösungsmittel- und Solutekonformere berücksichtigt werden. Dabei ist die Qualität der Vorhersageergebnisse unabhängig von der verwendeten gewichteten Mischung der Lösungsmittel- und Solutekonformere, die mit Hilfe von Konformeranalysen im Vakuum ermittelt wurden. Eine weitere Verbesserung der Vorhersagegenauigkeit kann erreicht werden, wenn die Konformere nicht im Vakuum sondern mit Methoden der Molekulardynamik in Gegenwart des gewünschten Lösungsmittels ermittelt werden. Die Oktanol-Wasser Verteilungskoeffizienten (log KOW) von Solutemolekülen verschiedener Stoffklassen konnten mit einem mittleren Fehler von 15% vorhergesagt werden. Diese Genauigkeit ist sehr gut und liegt im Bereich der bekannten Quantitative Structure – Activity Relationships. Die Vorteile des COSMO-RS Modells sind hier, dass eine Vorhersage auch für neuartige Wirkstoffe möglich ist, da ausschließlich die molekulare Struktur benötigt wird. Weiterhin wurde die Modellierung auf salzhaltige Lösungen erweitert, so dass es möglich ist, die Verteilungskoeffizienten in Gegenwart von chemischen Puffern bei verschiedenen pH- Werten vorherzusagen. Im zweiten Schritt wurde das entwickelte Vorgehen auf kolloidale Systeme angewendet. Als repräsentatives System wurden mizellare Lösungen ionischer und nichtionischer Tenside ausgewählt. Für mehrere Klassen organischer Substanzen wurden die Verteilungs-koeffizienten in verschiedenen mizellaren Lösungen (log KMW) mit einem mittleren Fehler von 7 bis 15 % vorhergesagt. Besonders hervorzuheben ist die Tatsache, dass auch ionische Substanzen (ionische Tenside, Salze) erfolgreich modelliert werden konnten. Insgesamt belegen die Ergebnisse, dass die a priori Vorhersage von Verteilungskoeffizienten komplexer Solutemoleküle, z.B. pharmazeutischer Wirkstoffe, mit dem COSMO-RS Modell möglich ist. Die erreichten Ergebnisse lassen erwarten, dass die erarbeitete Vorgehensweise auf andere kolloidale Systeme (z.B. Liposomen) und biologische Membranen übertragbar ist und somit eine universelle und zuverlässige Alternative zu systemspezifischen QSAR- Methoden darstellt.The present work deals with the modelling of partition coefficients of different solutes (including pharmaceuticals) in biological systems. The goal is to develop a reliable procedure to predict the partitioning of organic solutes between hydrophobic and hydrophilic phases. The considered hydrophobic phase may be of different complexity (octanol, micellar core, bilayer). The approach is based on the COSMO-RS model, which allows an a-priori prediction of the thermodynamic properties and thus the partition coefficients based only on the molecular structure of all system components. Therefore conformations of the molecular structure can be considered in the prediction. As a first step, the modelling approach has been exploited for the known system n-octanol/ water. The effect of the solute and solvent conformers has been studied. It has been shown, that single conformers of solute and solvent molecules have a remarkable influence on the predicted partition coefficients. The reliable prediction of partition coefficients is possible, when the weighted mixtures of solute and solvent conformers are considered in the modelling. In this case, the prediction quality is independent of the weighted mixture of the solute and solvent molecules identified by methods of conformational analysis in vacuum. Further improvement can be achieved when conformers are identified by molecular dynamics simulations in the presence of a solvent (n-octanol has been chosen as a representative solvent for the hydrophobic phase and water for the hydrophilic one). The n-octanol/water partition coefficients (log KOW) of different solutes classes as well as of a series of pharmaceuticals were predicted with an average error of 15%. It is considered to be a very good accuracy, since it lies within the typical errors of the accepted Quantitative Structure – Activity Relationships. The advantage of the COSMO-RS model is its ability to provide the a priori prediction of partition coefficients based on the molecular structure only that would be of special value for novel pharmaceuticals and active agents. Further on, the first calculations were made for the systems containing salts in the hydrophilic phase. The results show that the prediction of partitioning in the presence of buffers at different pH-values is principally possible. As a second step, the developed approach has been applied to the aggregated systems - aqueous micellar solutions. Partition coefficients (log KMW) of several solute classes in the systems containing non-ionic and ionic surfactants have been predicted with an average error of 7% and 15%, correspondingly. It has to be especially emphasized that the predictions in the systems containing ionic species such as ionic surfactants have been done successfully. The results demonstrate the applicability of the COSMO-RS model for the a priori prediction of partition coefficients of complex solutes, e.g., pharmaceuticals and active agents. Based on the presented results, we expect that the developed modelling approach can be also applied to the aggregated systems of higher complexity such as membranes or vesicles. Compared to the system specific QSAR methods, the COSMO-RS model can be considered as an universal and reliable alternative for the prediction of partition coefficients in the aggregated systems

Interdisciplinary characterization of T cell dynamics in HIV infection

HIV has caused one of the most devastating pandemics in modern medicine. HIV infects and kills on of the central players in effector immunity, CD4+ T cells, that provide helper mechanisms to all arms of the immune system. Although the virus indirectly affects most cells of the immune system, CD4+ and CD8+ T cells in particular become highly dysfunctional and show traits of severe immune pathology during the infection. These cells are of importance in adaptive immunity and recognize through their T cell receptors foreign antigens that are presented on MHC molecules. In the absence of normal T cell dynamics and homeostasis, host effector immunity collapses and most individuals develop AIDS, without antiretroviral therapy. The growing number of immunological variables measured today poses challenges to studying T cell dynamics in HIV infection. However, with the introduction of new techniques within bioinformatics, we now possess statistical tools to analyse combined measurements of T cell pathology, epitope targeting and dysfunction in the context of HIV infection. In all of these studies, multi-parametric flow cytometry and advanced bioinformatics were thus combined to study traits of T cell dynamics in HIV infection. By examining a broad range of T cell markers, we concluded in paper I that the CD4/CD8 ratio correlated with a significantly increased number of pathological T cell populations and was associated with CD4 recovery 2 years after ART initiation. These data indicate that the CD4/CD8 ratio would be a suitable clinical predictor of combined T cell pathology in HIV infection. By developing a novel epitope selection algorithm in paper II, we aimed to identify optimal MHC class II-restricted HIV epitopes with broad viral and host coverage. Employing both immunological and virological approaches, a set of peptides was shown to induce broad HIV-specific CD4+ T cell responses, where the number of targeted Gag epitopes was inversely correlated with HIV viral load. In order to further trace events of HIV disease progression, we investigated whether the combined pattern of HIV evolution and CD8+ T cell functionality could explain the risk of HIV disease progression in HLA-B*5701+ patients (paper III). HIV Gag sequence diversity was shown to be lower and multi-functional responses higher against wild-type and autologous HLA-B*5701-restricted epitopes in subjects of low risk of disease progression. Both of these studies highlight the power of multidisciplinary approaches, integrating complex evolutionary and immunological data, to understand the mechanisms underlying T cell dysfunction and pathogenesis. To further clarify why HIV-specific CD8+ T cells exhibit severe dysfunctionalcharacteristics in both treated and untreated HIV infection, we studied in paper IV the role of two central T-box transcription factors (T-bet and Eomes) using combined flow cytometry and bioinformatics. It was shown that HIV-specific CD8+ T cells almost exclusively have highly elevated levels of Eomes, but lower T-bet expression, which is associated with up-regulation of numerous inhibitory receptors, impaired functional characteristics and a transitional memory differentiation status. Surprisingly, these features were retrained despite any years on ART, implicating that the relationship between T-bet and Eomes might partly explain the inability of CD8+ T cells to control viral rebound post ART cessation. In summary, this thesis has combined the knowledge of immunology andvirology with the help of bioinformatics to study T cell dynamics in HIV infection. This interdisciplinary approach has increased our knowledge of factors that are linkedto T cell pathology, risk of disease progression and impaired T cell functionality

Perturbed CD8+ T cell TIGIT/CD226/PVR axis despite early initiation of antiretroviral treatment in HIV infected individuals.

HIV-specific CD8+ T cells demonstrate an exhausted phenotype associated with increased expression of inhibitory receptors, decreased functional capacity, and a skewed transcriptional profile, which are only partially restored by antiretroviral treatment (ART). Expression levels of the inhibitory receptor, T cell immunoglobulin and ITIM domain (TIGIT), the co-stimulatory receptor CD226 and their ligand PVR are altered in viral infections and cancer. However, the extent to which the TIGIT/CD226/PVR-axis is affected by HIV-infection has not been characterized. Here, we report that TIGIT expression increased over time despite early initiation of ART. HIV-specific CD8+ T cells were almost exclusively TIGIT+, had an inverse expression of the transcription factors T-bet and Eomes and co-expressed PD-1, CD160 and 2B4. HIV-specific TIGIThi cells were negatively correlated with polyfunctionality and displayed a diminished expression of CD226. Furthermore, expression of PVR was increased on CD4+ T cells, especially T follicular helper (Tfh) cells, in HIV-infected lymph nodes. These results depict a skewing of the TIGIT/CD226 axis from CD226 co-stimulation towards TIGIT-mediated inhibition of CD8+ T cells, despite early ART. These findings highlight the importance of the TIGIT/CD226/PVR axis as an immune checkpoint barrier that could hinder future "cure" strategies requiring potent HIV-specific CD8+ T cells

Do reduced numbers of plasmacytoid dendritic cells contribute to the aggressive clinical course of COVID-19 in chronic lymphocytic leukaemia?

Infections with SARS-CoV-2 have been unduly severe in patients with haematological malignancies, in particular in those with chronic lymphocytic leukaemia (CLL). Based on a series of observations, we propose that an underlying mechanism for the aggressive clinical course of COVID-19 in CLL is a paucity of plasmacytoid dendritic cells (pDCs) in these patients. Indeed, pDCs express Toll-like receptor 7 (TLR7), which together with interferon-regulatory factor 7 (IRF7), enables pDCs to produce large amounts of type I interferons, essential for combating COVID-19. Treatment of CLL with Bruton's tyrosine kinase (BTK) inhibitors increased the number of pDCs, likely secondarily to the reduction in the tumour burden.publishedVersio

Characterization of HIV-Specific CD4+T Cell Responses against Peptides Selected with Broad Population and Pathogen Coverage

CD4+ T cells orchestrate immunity against viral infections, but their importance in HIV infection remains controversial. Nevertheless, comprehensive studies have associated increase in breadth and functional characteristics of HIV-specific CD4+ T cells with decreased viral load. A major challenge for the identification of HIV-specific CD4+ T cells targeting broadly reactive epitopes in populations with diverse ethnic background stems from the vast genomic variation of HIV and the diversity of the host cellular immune system. Here, we describe a novel epitope selection strategy, PopCover, that aims to resolve this challenge, and identify a set of potential HLA class II-restricted HIV epitopes that in concert will provide optimal viral and host coverage. Using this selection strategy, we identified 64 putative epitopes (peptides) located in the Gag, Nef, Env, Pol and Tat protein regions of HIV. In total, 73% of the predicted peptides were found to induce HIV-specific CD4+ T cell responses. The Gag and Nef peptides induced most responses. The vast majority of the peptides (93%) had predicted restriction to the patient’s HLA alleles. Interestingly, the viral load in viremic patients was inversely correlated to the number of targeted Gag peptides. In addition, the predicted Gag peptides were found to induce broader polyfunctional CD4+ T cell responses compared to the commonly used Gag-p55 peptide pool. These results demonstrate the power of the PopCover method for the identification of broadly recognized HLA class II-restricted epitopes. All together, selection strategies, such as PopCover, might with success be used for the evaluation of antigen-specific CD4+ T cell responses and design of future vaccines

T-bet and Eomes Are Differentially Linked to the Exhausted Phenotype of CD8+T Cells in HIV Infection

CD8+ T cell exhaustion represents a major hallmark of chronic HIV infection. Two key transcription factors governing CD8+ T cell differentiation, T-bet and Eomesodermin (Eomes), have previously been shown in mice to differentially regulate T cell exhaustion in part through direct modulation of PD-1. Here, we examined the relationship between these transcription factors and the expression of several inhibitory receptors (PD-1, CD160, and 2B4), functional characteristics and memory differentiation of CD8+ T cells in chronic and treated HIV infection. The expression of PD-1, CD160, and 2B4 on total CD8+ T cells was elevated in chronically infected individuals and highly associated with a T-betdimEomeshi expressional profile. Interestingly, both resting and activated HIV-specific CD8+ T cells in chronic infection were almost exclusively T-betdimEomeshi cells, while CMV-specific CD8+ T cells displayed a balanced expression pattern of T-bet and Eomes. The T-betdimEomeshi virus-specific CD8+ T cells did not show features of terminal differentiation, but rather a transitional memory phenotype with poor polyfunctional (effector) characteristics. The transitional and exhausted phenotype of HIV-specific CD8+ T cells was longitudinally related to persistent Eomes expression after antiretroviral therapy (ART) initiation. Strikingly, these characteristics remained stable up to 10 years after ART initiation. This study supports the concept that poor human viral-specific CD8+ T cell functionality is due to an inverse expression balance between T-bet and Eomes, which is not reversed despite long-term viral control through ART. These results aid to explain the inability of HIV-specific CD8+ T cells to control the viral replication post-ART cessation

The Immune Synapses Reveal Aberrant Functions of CD8 T Cells During Chronic HIV Infection

Chronic HIV infection causes persistent low-grade inflammation that induces premature aging of the immune system including senescence of memory and effector CD8 T cells. To uncover the reasons of gradually diminished potency of CD8 T cells from people living with HIV, here we expose the T cells to planar lipid bilayers containing ligands for T-cell receptor and a T-cell integrins and analyze the cellular morphology, dynamics of synaptic interface formation and patterns of the cellular degranulation. We find a large fraction of phenotypically naive T cells from chronically infected people are capable to form mature synapse with focused degranulation, a signature of a differentiated T cells. Further, differentiation of aberrant naive T cells may lead to the development of anomalous effector T cells undermining their capacity to control HIV and other pathogens that could be contained otherwise

Functional SARS-CoV-2 cross-reactive CD4+ T cells established in early childhood decline with age.

Pre-existing SARS-CoV-2-reactive T cells have been identified in SARS-CoV-2-unexposed individuals, potentially modulating COVID-19 and vaccination outcomes. Here, we provide evidence that functional cross-reactive memory CD4+ T cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is established in early childhood, mirroring early seroconversion with seasonal human coronavirus OC43. Humoral and cellular immune responses against OC43 and SARS-CoV-2 were assessed in SARS-CoV-2-unexposed children (paired samples at age two and six) and adults (age 26 to 83). Pre-existing SARS-CoV-2-reactive CD4+ T cell responses targeting spike, nucleocapsid, and membrane were closely linked to the frequency of OC43-specific memory CD4+ T cells in childhood. The functional quality of the cross-reactive memory CD4+ T cell responses targeting SARS-CoV-2 spike, but not nucleocapsid, paralleled OC43-specific T cell responses. OC43-specific antibodies were prevalent already at age two. However, they did not increase further with age, contrasting with the antibody magnitudes against HKU1 (β-coronavirus), 229E and NL63 (α-coronaviruses), rhinovirus, Epstein-Barr virus (EBV), and influenza virus, which increased after age two. The quality of the memory CD4+ T cell responses peaked at age six and subsequently declined with age, with diminished expression of interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF), and CD38 in late adulthood. Age-dependent qualitative differences in the pre-existing SARS-CoV-2-reactive T cell responses may reflect the ability of the host to control coronavirus infections and respond to vaccination