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

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 the longitudinal HIV-1 quasispecies evolution in HIV-1 infected individuals co-infected with Mycobacterium tuberculosis

One of the earliest and most striking observations made about HIV is the extensive genetic variation that the virus has within individual hosts, particularly in the hypervariable regions of the env gene which is divided into 5 variable regions (V1-V5) and 5 more constant (C1-C5) regions. HIV evolves at any time over the course of an individual’s infection and infected individuals harbours a population of genetically related but non-identical viruses that are under constant change and ready to adapt to changes in their environment. These genetically heterogeneous populations of closely related genomes are called quasispecies [65]. Tuberculosis or tubercle forming disease is an acute and/or chronic bacterial infection that primarily attacks the lungs, but which may also affect the kidneys, bones, lymph nodes, and brain. The disease is caused by Mycobacterium tuberculosis (MTB), a slow growing rod-shaped, acid fast bacterium. It is transmitted from person to person through inhalation of bacteria-carrying air droplets. Worldwide, one person out of three is infected with Mycobacterium tuberculosis – two billion people in total. TB currently holds the seventh place in the global ranking of causes of death [73]. In 2008, there were an estimated 9.4 (range, 8.9–9.9 million) million incident cases (equivalent to 139 cases per 100 000 population) of TB globally [75]. A complex biological interplay occurs between M. tuberculosis and HIV in coinfected host that results in the worsening of both pathologies. HIV promotes progression of M. tuberculosis either by endogenous reactivation or exogenous reinfection [77, 78] and, the course of HIV-1 infection is accelerated subsequent to the development of TB [80]. Active TB is associated with an increase in intra-patient HIV-1 diversity both systemically and at the infected lung sites [64,122]. The sustainability or reversal of the HIV-1 quasispecies heterogeneity after TB treatment is not known. Tetanus toxoid vaccinated HIV-1 infected patients developed a transient increase in HIV-1 heterogeneity which was reversed after few weeks [121]. Emergence of a heterogeneous HIV-1 population within a patient may be one of the mechanisms to escape strong immune or drug pressure [65,128]. The existence of better fitting and/or immune escape HIV-variants can lead to an increase in HIV-1 replication [129,130]. It might be that TB favourably selected HIV-1 variants which are sources for consistent HIV-1 replication. Understanding the mechanisms underlying the impacts of TB on HIV-1 is essential for the development of effective measures to reduce TB related morbidity and mortality in HIV-1 infected individuals. In the present study we studied whether the increase in HIV-1 quasispecies diversity during active TB is reversed or preserved throughout the course of antituberculous chemotherapy. For this purpose Two time point HIV-1 quasispecies were evaluated by comparing HIV-1 infected patients with active tuberculosis (HIV-1/TB) and HIV-1 infected patients without tuberculosis (HIV-1/non TB). Plasma samples were obtained from the Frankfurt HIV cohort and HIV-1 RNA was isolated. C2V5 env was amplified by PCR and molecular cloning was performed. Eight to twenty five clones were sequenced from each patient. Various phylogenetic analyses were performed including tree inferences, intra-patient viral diversity and divergence, selective pressure, co-receptor usage prediction and two time point identity of quasispecies comparison using Mantel’s test. We found out from this study that: 1) Active TB sustains HIV-1 quasispecies diversity for longer period 2. Active TB increases the rate of HIV-1 divergence 3) TB might slow down evolution of X4 variants And we concluded that active TB has an impact on HIV-1 viral diversity and divergence over time. The influence of active TB on longitudinal evolution of HIV- 1 may be predominant for R5 viruses. The use of CCR5-coreceptor inhibitors for HIV-1/TB patients as therapeutic approach needs further investigation.Eine der ersten und überraschenden Beobachtungen, welche bei der Analyse des HI-Virus gemacht wurden ist seine ausgeprägte Genetische Variabilität besonders die hypervariable Region des env Genes betreffen. Dieses wird in 5 variable Regionen (V1-V5) sowie 5 stärker konservierte Regionen (C1-C5) unterteilt. HIV wandelt sich zu jedem Zeitpunkt im Verlauf der Infektion und jedes infizierte Individuum ist Träger einer Population von genetisch verwandten jedoch nicht identischen Viren, welche sich kontinuierlich verändern und an die Erfordernisse innerhalb der Umgebung anpassen. Diese genetisch heterogenen, jedoch eng verwandten Populationen werden Quasispecies genannt. Tuberkulose ist eine mykobakterielle Infektion, welche sowohl akute als auch chronische Verläufe zeigt. Neben den Lungen als primärem Manifestationsort können auch die Nieren, Knochen und andere Organe befallen sein. Eine von drei Personen weltweit ist mit Mycobacterium tuberculosis infiziert, insgesamt 2 Milliarden Menschen. In HIV/TB Co-Inifzierten Menschen entsteht ein komplexes Zusammenspiel zwischen HIV und M. tuberculosis, welches zu einer Verschlechterung beider Krankheitsbilder führt. HIV führt durch endogene Rekativierung oder exogene Re-Infektion zu einer Progression der Tuberkulose, welche im weiteren Verlauf die Krankheitsprogression von HIV beschleunigt. Sowohl Morbidität als auch Mortalität sind in HIV-1/TB Co-Infizierten Menschen erhöht. Aktive Lungentuberkulose und Miliartuberkulose gehen mit dem Anstieg der Diversifität der HIV Viren innerhalb eines Wirtes einher. Wie lange diese erhöhte Heterogenität der HIV Quasispecies nach der erfolgreichen Behandlung einer Tuberkulose bestehen bleibt ist bisher noch unklar. Das Verständnis des dem Zusammenspiel von HIV und TB zugrundeliegenden Mechanismus ist essentiell für die Entwicklung von effektiven Massnahmen zur Senkung der Morbidität und Mortalität in HIV/TB Co-infizierten Menschen. Die gegenwärtige Forschungsarbeit folgte daher der Frage, ob wärend einer aktiven TB Infektion eine Zunahme der Diversität der HIV-1 Quasispecies zu beobachten ist und ob diese Diversität während einer TB Therapie erhalten bleibt oder sich zurück bildet. Hierfür wurden die HIV-1 Quasispecies zu zwei Zeitpunkten untersucht, wobei Proben von HIV-1 infizierten Patienten mit aktiver Tuberkulose (HIV-1/TB) und HIV infizierte Patienten ohne Tuberkulose (HIV-1/non TB) verglichen wurden. Aus Plasmaproben der Frankfurter HIV Cohorte wurde HIV-1 RNA isoliert. C2V5 env wurde durch PCR amplifiziert und molekular cloniert. Acht bis fünfundzwanzig Clone wurden für jeden Patienten sequenziert. Mehrere phylogenetische Analysen wurden durchgeführt, welche tree inferences, Intra-Patienten- und virale Diversität und Divergenz, Selektionsdruckanalysen, Vorhersage der Co-Rezeptornutzung sowie Zweipunktanalysen der Identität von Quasispecies mit Hilfe des Mantel’s Test miteinschlossen. Die Analysen ergaben die folgenden Ergebnisse: 1) Eine aktive TB erhält die Diversität von HIV-1 Quasispecies über einen längeren Zeitraum. 2. Eine aktive TB verstärkt die HIV -1 Divergenz 3) TB könnte zu einer langsameren Evolution von X4 Varianten führen. Schlussfolgerung: eine aktive TB beeinflusst die Entwicklung der viralen Diversität und Divergenz von HIV-1 im Verlauf der Krankheit. Der Einfluss der aktiven TB auf die longitudinale Evolution von HIV-1 könnte insbesondere R5 Viren betreffen. Der Einsatz von CCR5-Corezeptor Inhibitoren in HIV-1/TB coinifizerten Patienten sollte daher in Langzeitstudien untersucht werden

Generation of enhanced gene delivery vectors by directed evolution of adeno-associated virus

Despite promising advance in the development of viral vectors based on AAV for human gene therapy, several major hurdles for a more general use remain. Among these, efficient in vivo applications are limited by the high prevalence of neutralizing antibodies in the human population, which can reduce or eliminate transgene expression. A successful prevention of antibody-mediated vector neutralisation requires the modification of specific epitopes of the viral capsid responsible for Ab binding. The aim of this work was to demonstrate that immune-escaping capsid variants can be generated through genetic modifications of the virus by taking advantage of combinatorial engineering and directed evolution protocols. A library of 107 AAV mutants carrying random point mutations scattered throughout the capsid gene of AAV was created by error prone PCR and screened for clones that were able to avoid neutralization by AAV-neutralizing human sera. Three mutants carrying the mutations R459G, R459K and N551D respectively and a double mutant with a combined R459K/N551D mutation were strongly enriched after the selection procedure. Characterisation of these clones showed an immune-escaping phenotype for all mutants. However, the combination mutant proved to be superior in both evasion of neutralization and infectivity, leading to the assumption that multiple mutations convey enhanced effects. Therefore, the remaining pool was subjected to DNA shuffling and additional error prone PCR, yielding a second-generation library, which was screened for further improved phenotypes. In this context, a method which we called evolution monitoring was devised allowing optimization of several experimental conditions that are typically critical for successful outcome of library panning. These refinements yielded novel variants with further enhanced immune-escape abilities and infectivity in comparison to previously selected mutants. Finally, obtained data suggests an enormous potential for using the here developed tools to study infection biology of viruses by reverse genetics. This work showed for the first time that error prone PCR and DNA shuffling can be successfully applied for genetic engineering of a virus by a directed evolution approach. In principle, using appropriate selection protocols these techniques should be adaptable for addressing a wide variety of challenges concerning AAV in particular and virology in general

Computational approaches for improving treatment and prevention of viral infections

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

Analysis of HIV-host interaction on different scales

The human immunodeficiency virus depends on molecular pathways of the host for efficient replication and spread. The intricate network of host-virus interactions shapes the virus\u27; evolution by driving the pathogen to evade immune recognition and constraining it to maintain its capacity to replicate. Study of the HIV-host interactions provides important insights into viral evolution, pathogenicity and potential treatment strategies. This thesis presents an analysis of HIV-host interactions on several scales, ranging from individual protein interactions to whole genomes. On the scale of individual interaction we analyze structural and physical determinants of the interaction between host TRIM5alpha and virus capsid — an interaction of potential therapeutic interest due to the capacity of TRIM5alpha to block retroviral infections. On the scale of viral population we present two studies of a highly variable region of the virus genome involved in the interaction with host cell coreceptors upon virus cell entry. The studies provide insights into the virus evolution and the physicochemical and structural properties related to its interaction with cellular coreceptors. On the scale of the single cell we develop models of HIV cell entry involving virus, host and environmental factors. The models represent a comprehensive picture of the virus phenotype and allow one to view the variability of virus phenotypes on 2D phenotype maps. On the genomic scale we perform a large-scale analysis of all HIV-host interactions. This study reveals insights into general patterns of the host-pathogen evolution and suggests candidate host proteins involved in interactions potentially important for the infection and interesting for further study on other scales. Interactions and processes crucial for the HIV infection reemerge across the scales pointing to the importance of integrative, multi-scale studies of host-pathogen biology.Das Humane Immundefizienz-Virus hängt von molekularen Mechanismen des Wirts für seine effiziente Replikation und Ausbreitung ab. Das komplizierte Netzwerk von Wirt-Virus Interaktionen formt die Evolution des Virus, indem es den Erreger dazu bringt, sich der Erkennung durch das Immunsystem zu entziehen und seine Replikationskapazität aufrecht zu erhalten. Das Studium der HIV-Wirt Interaktionen erlaubt wichtige Einblicke in die viralen Evolution, die Pathogenität des Virus, sowie mögliche Behandlungsstrategien. Diese Arbeit stellt eine Analyse der HIV-Wirt-Interaktionen in mehreren Größenordnungen vor, von einzelnen Protein-Interaktionen bis hin zur Analyse ganzer Genome. In Hinblick auf einzelne Interaktionen untersuchen wir strukturelle und physikalische Determinanten der Interaktion zwischen dem Wirtfaktor TRIM5alpha; und dem viralen Kapsid - eine Interaktion, die von therapeutischem Interesse ist wegen der Fähigkeit von TRIM5alpha, retrovirale Infektionen zu blockieren. In Hinblick auf virale Populationen präsentieren wir zwei Studien einer hochvariablen Region des viralen Genoms, die in der Interaktion des Virus mit zellulären Rezeptoren des Wirts beim viralen Zelleintritt involviert sind. Diese Studien geben Einblick in die virale Evolution und die physikalisch-chemischen und strukturellen Eigenschaften des Virus bezüglich dessen Interaktion mit zellulären Ko-Rezeptoren. Auf der Skala der einzelnen Zelle entwickeln wir Modelle des HIV Zelleintritts welche das Virus, den Wirt und Umgebungsfaktoren berücksichtigen. Diese Modelle bieten ein umfassendes Bild des viralen Phänotyps und erlauben es, die Variabilität des Virus auf 2D-Phänotyp-Karten zu visualisieren. Im genomweiten Maßstab führen wir eine groß angelegte Analyse aller HIV-Wirt-Interaktionen durch. Diese Studie erlaubt Einblicke in allgemeine Muster der Wirt-Pathogen-Evolution und identifiziert Kandidaten für Wirtsproteine, deren Interaktionen potenziell wichtig für die virale Infektion sind und deren weitere Untersuchung in anderen Größenordnungen von Interesse ist. Interaktionen und Prozesse, die von entscheidender Bedeutung für die HIV-Infektion sind zeigen sich wiederholt in allen untersuchten Maßstäben und unterstreichen die Bedeutung einer integrativen und multi-skalaren Untersuchung der Wirt-Pathogen-Biologie

Vorhersage der HIV-1 Co-Rezeptor Nutzung der Viralen Quasispecies mittels Classifier Ensembles

In this work a new approach to determine the co-receptor tropism of HIV patients is presented and evaluated against different type of clinical patient data. A profound knowledge of co-receptor tropism, the type host-cell co-receptor used for cell-entry by the virus present in a patient, is required for an effective treatment with novel entry inhibitors like Maraviroc. The structural basis for the fusion process of virus and host-cell seems to be the interaction of a short variable loop (V3) of virus protein gp120 with one of the host-cell co-receptors CCR5 or CXCR4. Based on V3 loop sequences of viruses with known tropism, machine learning methods have been trained and are often successful in correctly predicting the tropism from patient derived V3 sequences. These, so called, genotypic methods present a cost-efficient, fast and broadly available alternative to cell-based, phenotypic assays. The machine learning approach developed here, uses ensemble classifiers for an improved prediction performance based on two different properties derived from V3 loop sequences and their 3D structure. The subsequent application to viral quasispecies, the virus population ensemble found in a patient, extracted by next-generation sequencing (NGS), shows that a combination of NGS with genotypic tropism prediction can have great benefits compared to phenotypic testing, making it a desirable part of the diagnostic routine.Diese Arbeit stellt einen neuen Ansatz zur Bestimmung des korezeptor Tropismus von HIV Patienten vor und erprobt diese anhand verschiedener klinischer Patientendaten. Eine gesicherte Kenntnis des korezeptor Tropismuses, der Art des Wirtszellen-Korezeptors, welcher das Virus im Patienten für den Zelleintritt benutzt, ist nötig um eine effektive Behandlung mit neuen Eintrittsinhibitoren wie Maraviroc zu gewährleisten. Die strukturelle Grundlage für den Fusionsprozess des Virus mit der Wirtszelle scheint die Interaktion einer kurzen, variablen Schleife (V3) des Virusproteins gp120 mit einem der Wirtszell-Korezeptoren CCR5 oder CXCR4 zu sein. Basierend auf V3 Sequenzen mit bekanntem Tropismus wurden bereits Maschinen-Lern-Verfahren entwickelt, welche häufig eine korrekte Vorhersage über den Tropismus neuer V3 Sequenzen aus Patientenvirenstämmen treffen können. Diese, so genannten, genotypischen Methoden stellen eine preiswerte, schnelle und hoch verfügbare Alternative zu Zell-basierten, Phenotypischen Assays dar. Das hier vorgestellte Maschinen-Lern-Verfahren, verwendet Ensemble Classifier für eine verbesserte Vorhersage, basierend auf zwei unterschiedlichen Eigenschaften der V3 Sequenzen und 3D-Strukturen. Die anschließende Anwendung auf Virus-Quasispezies, das Virus Populationsensemble innerhalb eines Patienten, extrahiert durch Next-Generation-Sequencing (NGS), zeigt das die Kombination von NGS mit genotypischer Tropismus Vorhersage große Vorteile gegenüber phenotypischen Tests bietet diese zu einem wünschenswertem Teil der Routinediagnostik macht

Development of Microsphere-based Molecular and Serodiagnostics and Their Clinical Utility

Microbial infections share many symptoms in common, rendering diagnosis difficult solely on clinical grounds. Thus, rapid, cost-effective and reliable tests are necessary for the diagnosis of infectious diseases. While the traditional diagnosis is mostly confined to detection of one pathogen at a time, a multiplex array could be a feasible alternative to improve the efficiency in the detection of infections. The Luminex xMAP-based high-throughput platform can provide simultaneous analysis of multiple analytes from the same sample by utilising differentially dyed microspheres. In this thesis, I developed xMAP-based Suspension Immuno Assays (SIAs) for the determination of IgG antibodies, IgM antibodies, as well as the avidity of IgG against the human cytomegalovirus (HCMV), Toxoplasma gondii (T. gondii) or human parvovirus B19 (B19V). Moreover, I also developed xMAP-based multiplex DNA assays for 13 human polyomaviruses (HPyVs). Primary infections by HCMV, T. gondii and B19V during pregnancy can result in severe consequences to the foetus. The serological status of the mother is critically important in counselling and recognition of infections. Hereby, I developed and evaluated SIAs for IgG, IgM and IgG-avidity against these three important pathogens. Diagnostic performances of the new assays were assessed with more than 1000 well-characterised serum samples. All the newly developed assays exhibited excellent performance compared to corresponding high-quality reference methods. The positive and negative percent agreements of the antibody-SIAs in comparison with high-standard reference assays were 92-100% and 95-100%, respectively. Kappa efficiencies between SIAs and corresponding reference assays were 0.94-1. Intra-assay and inter-assay coefficients of variations ranged between 2-12% and 1-19%, respectively. Among clinical samples from individuals with primary infection, the IgM- and IgG-SIAs served as highly sensitive screening means for detection of acute infections and immune status; and IgG-avidity-SIAs as a highly specific confirmatory approach for separation of primary infections from long-term B-cell immunity. On the other hand, during the past 12 years, a dozen viruses have joined the known family members of HPyVs. Serological studies have shown that HPyV infections occur at young age and most of the viruses circulate widely in the general population. Although HPyV infections are generally asymptomatic, severe complications can arise due to virus reactivations in immunocompromised or elderly individuals. HPyVs can persist lifelong after primary infection; however, their tissue specificities, persistence sites and transmission routes are still unclear. Also, the clinical manifestations of HPyVs with regard to immune suppression are largely unidentified. To this end, I developed xMAP-based multiplex DNA assays for all 13 HPyVs known before 2017, by using primer pairs and probes targeting the respective HPyV major capsid protein VP1 genes. The xMAP-based multiplex assays allowed for simultaneous detection of all the HPyVs with detection limits of 1-100 copies/µl. At high copies (105 copies/µl) each of the 13 target sequences were identified correctly with no cross-reactions. With this novel and specific assay, the extent to which the lymphoid system plays a role in the HPyV infection and persistence was assessed. The frequency of occurrence of HPyV viral genomes was explored in 78 lymphoid tissues from children and adults with tonsillar diseases. HPyV-DNA was found in 17.9% (14/78) of tonsils: JC polyomavirus (JCPyV, n=1), WU polyomavirus (WUPyV, n=3), Merkel cell polyomavirus (MCPyV, n=1), human polyomavirus 6 (HPyV6, n=6), trichodysplasia spinulosa polyomavirus (TSPyV, n=3). The observed frequent occurrence of HPyVs in human tonsils suggests the lymphoid tissue plays an important role as a potential transmission route and a location of persistence for these viruses. However, whether or not the undetected HPyVs share the same infection route requires more investigation with different sample types. Furthermore, to determine the occurrences in skin and clinical associations of HPyVs, I studied their genoprevalences in biopsies of premalignant [squamous cell carcinoma in situ (SCCis) or actinic keratosis (AK)] lesional vs. benign skin from 126 liver transplant recipients (LiTRs); as well as in healthy skin of 80 immunocompetent adults. Multiplex screening was followed by singleplex qPCRs of positive samples, for reference and quantification of the viral DNAs. In total, five dermal HPyVs – MCPyV, HPyV6, human polyomavirus 7, TSPyV, and Lyon IARC polyomavirus (LIPyV) – were found in 26.2% (58/221) skin biopsies. The prevalences and quantities of MCPyV in premalignant vs. benign skin of LiTRs were similar to those in healthy skin of controls. TSPyV was found in a single skin lesion at very low copies. The other three HPyVs occurred exclusively in benign skin. Overall, in 10 out of 12 SCCis/AK patients the viral DNA findings in skin were alike. Thereby, the occurrences of HPyVs in the skin of LiTRs and controls speak against a role for any of HPyVs in SCC development. The work presented in this thesis shows that the xMAP-based serological approaches exhibit excellent diagnostic performances compared to corresponding conventional methods. Moreover, the developed xMAP-based multiplex PCR for 13 HPyVs could be applied successfully in a variety of clinical materials. Altogether, the newly developed systems provide a powerful tool for medical diagnosis and research.Tartuntatautien nimeäminen pelkästään kliinisen kuvan perusteella on haasteellista ja usein jopa mahdotonta. Tarvitaan luotettavia, nopeita ja kustannushyödyllisiä laboratoriomenetelmiä. Siinä missä perinteiset menetelmät ovat taudinaiheuttajakohtaisia, monikanavaisten ns. multiplex-menetelmien etuna on ylivoimainen tehokkuus ja kustannushyödyllisyys. Luminex xMAP -testialustalla voidaan kustakin potilasnäytteestä tehdä suuri joukko samanaikaisia määrityksiä perustuen erivärisiin mikrohelmiin. Väitöstyössä suunniteltiin ja pystytettiin xMAP -perustaisia ns. suspensio-immunomenetelmiä (SIA) mikrobikohtaisiin IgG-, IgM- ja IgG-aviditeetti (AVI) -määrityksiin, kahdelle virukselle ja yhdelle alkueläimelle: sytomegalovirus (HCMV), parvorokkovirus (B19V) ja toksoplasma (T. gondii). Niin ikään pystytettiin xMAP-perustainen multiplex-menetelmä 13:n ihmis-polyoomaviruksen (HPYV) nukleiinihappo (DNA) -diagnostiikkaan. Raskaudenaikaiset HCMV-, B19V ja T. gondii- ensitartunnat voivat vahingoittaa sikiötä. Koska odottavan äidin vasta-ainetulokset ovat hyvin tärkeitä kyseisten infektioiden ja niihin kohdistuvan immuniteetin määrityksessä, kehitimme ja evaluoimme IgG-, IgM- ja AVI-SIA-menetelmät näille kolmelle tärkeälle patogeenille. Uusien menetelmien suorituskyky selvitettiin >1000:n hyvinkarakterisoidun seeruminäytteen avulla. Menetelmien suorituskyky osoittautui erinomaiseksi korkeatasoisten vertailumenetelmien suhteen: Positiiviset ja negatiiviset yhtäpitävyysprosentit olivat 92-100% ja 95-100% (vast). Kappa-vakiot olivat 0.94-1. Testien sisäiset ja testien väliset variaatiokertoimet olivat 2-12% ja 1-19% (vast). IgM- ja IgG-SIA:t toimivat erittäin sensitiivisinä seulontamenetelminä akuutin infektion ja immuniteetin tunnistuksessa; ja AVI-SIA:t erittäin spesifisinä varmistusmenetelminä, erotettaessa k.o. ensitartuntoja vanhasta B-soluimmuniteetista. Toisaalta, viimeisten 12 vuoden aikana on löydetty 12 aiemmin tuntematonta ihmis-polyoomavirusta (HPyV). Polyoomavirusheimon jäsenille on tunnusomaista tartuntojen ajoittuminen nuoreen ikään ja virusten yleisyys väestössä. Vaikka infektiot useimmiten ovat oireettomia, näiden virusten uudelleen-aktivaatiolla immuunipuutteisissa tai iäkkäissä yksilöissä voi olla vakavia seurauksia. HPyV:t pesiytyvät elimistöön piilevässä muodossa koko eliniäksi; tämän persistenssin kudos- ja solutyypit ovat kuitenkin epäselvät, samoin kuin tartuntareitit. Puutteellisesti tunnettuja ovat myöskin HBoV:ien kliiniset ilmentymät immuunipuutoksien yhteydessä. Tämänlaisia tarkoitusperiä varten pystytimme xMAP-perustaiset multiplex-DNA-monistusmenetelmät kaikille 13:lle HPyV:lle (jotka tunnettiin 2017). Alukeparit ja koettimet suuntautuivat kussakin tapauksessa pääasiallisen kapsidiproteiinin VP1 geenialueelle. Aikaansaaduilla multiplex-menetelmillä kyettiin löytämään jokainen HPyV, detektiorajoilla 1-100 kopiota/ml. Korkeammissa pitoisuuksissa (105 geenikopiota/µl) identifioitui kukin 13:sta kohteesta ilman ristireaktioita. Tällä spesifisellä, uudella menetelmällä ryhdyimme ensialkuun selvittämään imukudoksen merkitystä HPyV-infektiossa ja -persistenssissä. Määritimme HPyV-genomien esiintyvyyden 78 (lasten, aikuisten) nielurisakudosnäytteessä. Kaikkinensa HPyV-DNA:ta löytyi 17,9% (14/78) tonsilloista, seuraavin lukumäärin: JCPyV (1), WUPyV (3), MCPyV (1), HPyV6 (6) TSPyV (3). Monilukuisuus ihmistonsilloissa viittaa imukudoksen oleelliseen rooliin kyseisten virusten tartunnoissa ja/tai persistenssissä. Löytymättä jääneiden HPyV:ien johtopäätökset riippuvat lisätutkimuksista. Seuraavaksi selvitimme HPyV:ien esiintyvyyttä ja kliinistä merkitystä ihokudoksessa. Tutkimuskohteina olivat koepalat ihon okasolusyövän esiasteista (ca in situ [SCCis] sekä aktiininen keratoosi [AK]). Näytteinä oli sekä sairaita että terveitä ihoalueita 126:n maksansiirtopotilaan seurannasta; ja vertailukohtana 80:n perusterveen henkilön tavanomainen iho. Multiplex-DNA-seulontatuloksia verrattiin viruskohtaisiiin (singleplex-formaatin) kvantitatiivisiin PCR-tuloksiin. Kaiken kaikkiaan, 221:stä ihobiopsiasta 58:sta (26,2%) löytyi viiden eri HPyV:n DNA:ta: MCPyV, HPyV6, HPyV7, TSPyV, LIPyV. Tulokset maksansiirtopotilaiden ihosyöpä-esiasteissa ja terveessä ihossa olivat samankaltaiset sekä keskenänsä että verrattuna terveeseen normaaliväestöön. Tuloksemme puhuvat voimakkaasti polyoomavirusten etiologista osuutta vastaan ihon okasolusyövän synnyssä. Tässä väitöstyössä todistetaan pystyttämämme xMAP-pohjaisen ”täsmäserologian” erinomainen suorituskyky verrattuna totunnaisiin diagnoosimenetelmiin. Toisaalta, uutta xMAP-multiplex-PCR-menetelmäämme käytetään menestyksellisesti 13:n ihmis-polyoomaviruksen DNA:n osoitukseen erityyppisissä potilasnäyteaineistoissa. Kaikkinensa, aikaansaatujen uusien tutkimusmenetelmiemme osoitetaan soveltuvan oivallisesti sekä lääketieteelliseen taudinmääritykseen että tieteentekoon

Predicting and analyzing HIV-1 adaptation to broadly neutralizing antibodies and the host immune system using machine learning

Thanks to its extraordinarily high mutation and replication rate, the human immunodeficiency virus type 1 (HIV-1) is able to rapidly adapt to the selection pressure imposed by the host immune system or antiretroviral drug exposure. With neither a cure nor a vaccine at hand, viral control is a major pillar in the combat of the HIV-1 pandemic. Without drug exposure, interindividual differences in viral control are partly influenced by host genetic factors like the human leukocyte antigen (HLA) system, and viral genetic factors like the predominant coreceptor usage of the virus. Thus, a close monitoring of the viral population within the patients and adjustments in the treatment regimens, as well as a continuous development of new drug components are indispensable measures to counteract the emergence of viral escape variants. To this end, a fast and accurate determination of the viral adaptation is essential for a successful treatment. This thesis is based upon four studies that aim to develop and apply statistical learning methods to (i) predict adaptation of the virus to broadly neutralizing antibodies (bNAbs), a promising new treatment option, (ii) advance antibody-mediated immunotherapy for clinical usage, and (iii) predict viral adaptation to the HLA system to further understand the switch in HIV-1 coreceptor usage. In total, this thesis comprises several statistical learning approaches to predict HIV-1 adaptation, thereby, enabling a better control of HIV-1 infections.Dank seiner außergewöhnlich hohen Mutations- und Replikationsrate ist das humane Immundefizienzvirus Typ 1 (HIV-1) in der Lage sich schnell an den vom Immunsystem des Wirtes oder durch die antiretrovirale Arzneimittelexposition ausgeübten Selektionsdruck anzupassen. Da weder ein Heilmittel noch ein Impfstoff verfügbar sind, ist die Viruskontrolle eine wichtige Säule im Kampf gegen die HIV-1-Pandemie. Ohne Arzneimittelexposition werden interindividuelle Unterschiede in der Viruskontrolle teilweise durch genetische Faktoren des Wirts wie das humane Leukozytenantigensystem (HLA) und virale genetische Faktoren wie die vorherrschende Korezeptornutzung des Virus beeinflusst. Eine genaue Überwachung der Viruspopulation innerhalb des Patienten, gegebenfalls Anpassungen der Behandlungsschemata sowie eine kontinuierliche Entwicklung neuer Wirkstoffkomponenten sind daher unerlässliche Maßnahmen, um dem Auftreten viraler Fluchtvarianten entgegenzuwirken. Für eine erfolgreiche Behandlung ist eine schnelle und genaue Bestimmung der Anpassung einer Variante essentiell. Die Thesis basiert auf vier Studien, deren Ziel es ist statistische Lernverfahren zu entwickeln und anzuwenden, um (1) die Anpassung von HIV-1 an breit neutralisierende Antikörper, eine neuartige vielversprechende Therapieoption, vorherzusagen, (2) den Einsatz von Antikörper-basierte Immuntherapien für den klinischen Einsatz voranzutreiben, und (3) die virale Anpassung von HIV-1 an das HLA-System vorherzusagen, um den Wechsel der HIV-1 Korezeptornutzung besser zu verstehen. Zusammenfassend umfasst diese Thesis mehrere statistische Lernverfahrenansätze, um HIV Anpassung vorherzusagen, wodurch eine bessere Kontrolle von HIV-1 Infektionen ermöglicht wird

Cell and organoid models to develop new antivirals and diagnostic methods for emerging viruses

The ongoing global pandemic of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) is only the latest reminder of the urgent need for new antiviral strategies and diagnostic tools to improve preparedness for emergence of new pathogenic RNA-viruses. A key obstacle for the development of antivirals and diagnostic tools has been the lack of good models to study RNA viruses. This thesis focused on cellular and organoid models for virus infection to facilitate the development of host-targeting antivirals and new diagnostic tools against emerging viruses. In Paper I, two close analogues from an in-house library of 425 host-targeting compounds were found to have antiviral activity against apathogenic RNA virus Hazara virus (HAZV). These two hit compounds were discovered using a cell- and image-based phenotypic antiviral screening assay. Subsequently, the two compounds were confirmed to have a broad-spectrum antiviral activity against several pathogenic RNA viruses including SARS-CoV-2, Ebola and Crimean-Congo hemorrhagic fever virus. After showing the independence of the compound’s antiviral effect of their originally designed target, human 8-oxoguanine glycosylase 1, thermal protein profiling was used to study the compound target. Applying this technique, the compound was revealed to disturb proteostasis pathways and interactions between cellular heat shock protein 70 complex and viral proteins. In Paper II, a second antiviral screening of in-house compounds was performed using the previously established image-based phenotypic antiviral screening assay with Zika virus (ZIKV) infected cells. The broad-spectrum activity of the compounds was confirmed by revealing the same compounds to be active against both HAZV and ZIKV. Next, the therapeutic window and antiviral activity of the top-hit compounds was demonstrated in several cellular models of ZIKV infection. Additionally, the novel antiviral compounds showed an antiviral effect and reversed ZIKV induced neurotoxicity in iPS cell derived human brain organoids. Furthermore, mechanism-of-action studies revealed the compound to impair the formation of new virus particles in the late lifecycle steps. In Paper III, the development of a new diagnostic tool for ZIKV is described using in vitro infected U87 cells and PBMCs. In the method presented, ZIKV cDNA was hybridized using padlock probes and amplified by two rounds of Circle-to-Circle Amplification. Detection was performed using a microfluidic affinity chromatography enrichment platform. Benchmarking of the newly developed method against RT-qPCR, the gold standard diagnostic method for ZIKV detection, confirmed a good correlation between both methods. Altogether, this thesis demonstrates how cell culture tools with varying complexity, unique advantages and challenges can be used to augment the development of novel antiviral drugs and diagnostic methods

Principles of Massively Parallel Sequencing for Engineering and Characterizing Gene Delivery

The advent of massively parallel sequencing and synthesis technologies have ushered in a new paradigm of biology, where high throughput screening of billions of nucleid acid molecules and production of libraries of millions of genetic mutants are now routine in labs and clinics. During my Ph.D., I worked to develop data analysis and experimental methods that take advantage of the scale of this data, while making the minimal assumptions necessary for deriving value from their application. My Ph.D. work began with the development of software and principles for analyzing deep mutational scanning data of libraries of engineered AAV capsids. By looking at not only the top variant in a round of directed evolution, but instead a broad distribution of the variants and their phenotypes, we were able to identify AAV variants with enhanced ability to transduce specific cells in the brain after intravenous injection. I then shifted to better understand the phenotypic profile of these engineered variants. To that end, I turned to single-cell RNA sequencing to seek to identify, with high resolution, the delivery profile of these variants in all cell types present in the cortex of a mouse brain. I began by developing infrastructure and tools for dealing with the data analysis demands of these experiments. Then, by delivering an engineered variant to the animal, I was able to use the single-cell RNA sequencing profile, coupled with a sequencing readout of the delivered genetic cargo present in each cell type, to define the variant’s tropism across the full spectrum of cell types in a single step. To increase the throughput of this experimental paradigm, I then worked to develop a multiplexing strategy for delivering up to 7 engineered variants in a single animal, and obtain the same high resolution readout for each variant in a single experiment. Finally, to take a step towards translation to human diagnostics, I leveraged the tools I built for scaling single-cell RNA sequencing studies and worked to develop a protocol for obtaining single-cell immune profiles of low volumes of self-collected blood. This study enabled repeat sampling in a short period of time, and revealed an incredible richness in individual variability and time-of-day dependence of human immune gene expression. Together, my Ph.D. work provides strategies for employing massively parallel sequencing and synthesis for new biological applications, and builds towards a future paradigm where personalized, high-resolution sequencing might be coupled with modular, customized gene therapy delivery.</p