In the present study, we have approached the molecular characterization of the HCMV specific UL1. To this end a HCMV (AD169-derived HB5 background) recombinant with an HA-epitope tagged UL1 and a mutant with a full UL1 deletion in the endotheliotropic HCMV TB40/E strain were generated. Our data reveal that the UL1 is transcribed with late kinetics. pUL1 is glycosylated and localizes at the site of virus assembly and secondary envelopment in infected cells forming part of the envelope of HCMV virions. A HCMV mutant with a targeted deletion of UL1 exhibits a growth defect phenotype in retinal pigment epithelium cells but not in fibroblasts, indicating that this ORF encodes a cell-type specific tropism factor. / En aquest treball hem investigat la pauta oberta de lectura de UL1 del Cytomegalovirus humà (HCMV), el gen UL1 es específic del HCMV. Hem caracteritzat la proteïna UL1 modificada amb un epítop HA en la soca HB5, derivada de AD169. L'UL1 s’expressa com una glicoproteïna que es pot detectar a les 48 i 72h post-infecció. En fibroblasts humans infectats, UL1 co-localitza al citoplasma, al lloc d’assemblatge del virió, amb proteïnes estructurals del virus. A més a més, els anàlisis de virions AD169 purificats que contenen UL1-HA mostren que UL1 és un nou constituent de l’envolta del HCMV. La delecció de UL1 en el context de la soca TB40/E del HCMV disminueix el creixement viral de manera selectiva en determinats tipus cel•lulars, suggerint que UL1 podria estar involucrat en la regulació del tropisme cel•lular del HCMV.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Orientador: Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T09:16:08Z (GMT). No. of bitstreams: 1 Andrade_PaulaDurante_M.pdf: 3994483 bytes, checksum: eca47ce9f7df65d68a6da3ad72ad056b (MD5) Previous issue date: 2009 / Resumo: O Citomegalovírus Humano (HCMV) é o principal causador de complicações pós-transplante. Métodos específicos que permitam identificar, precocemente, os pacientes com risco de desenvolvimento de doença, para os quais tratamento é indicado, têm sido requeridos, a fim de que poucos pacientes sejam desnecessariamente tratados e para a efetiva instituição terapêutica. A "Nested-PCR", dupla Reação em Cadeia da Polimerase, é um teste comumente utilizado no diagnóstico da infecção ativa por HCMV, contudo, quando realizada em leucócitos do sangue periférico, devido à sua alta sensibilidade, não apresenta boa correlação com o desenvolvimento de doença por HCMV. A detecção do DNA do HCMV no soro, pela PCR, tem sido associada com o desenvolvimento de doença por HCMV. Neste estudo, nós aplicamos a "Nested-PCR" em leucócitos do sangue periférico (denominada "L-PCR"), o método Convencional do Laboratório, e em soro ("sPCR"), para o diagnóstico da infecção ativa por HCMV, a fim de estabelecermos a correlação dos resultados obtidos, de ambos os métodos, com o desenvolvimento de infecção sintomática. Com este propósito, nós avaliamos, prospectivamente, amostras de 37 pacientes, 20 submetidos a transplante renal, e 17 submetidos a transplante de células tronco hematopoiéticas. Para excluir resultados falso negativos, na reação de amplificação pela "sPCR", um controle interno foi construído e todas as reações foram realizadas utilizando-o. De acordo com os critérios estabelecidos neste estudo, 21 pacientes (21/37 - 57%) desenvolveram infecção ativa por HCMV. Todos os pacientes com infecção ativa por HCMV foram positivos para a "L-PCR" (p= 0,0003). A "sPCR" foi positiva para somente 10 pacientes (10/37 - 27%) (p= 0,01). Resultados discordantes foram observados em 11 pacientes que foram positivos para infecção ativa para a "L-PCR", mas negativos para a "sPCR", 5 dos quais desenvolveram sintomas clínicos para o HCMV. O coeficiente Kappa de concordância observado para ambos os métodos foi de 0,44 (acordo moderado). Em dezesseis pacientes (43%), dos 37 estudados, não foram observados resultados positivos por nenhum dos métodos empregados - não desenvolveram infecção ativa -, contudo, 2 destes desenvolveram sintomas clínicos de provável doença por HCMV e um apresentou, posteriormente, biópsia confirmativa para doença por HCMV. Sintomas clínicos foram observados em 14 pacientes, em 12 deles infecção ativa foi diagnosticada através da "L-PCR" (p= 0,007) e, em 7, através da "sPCR" (p= 0,02). Ausência de sintomas clínicos foram observados em 9 pacientes nos quais a "L-PCR" detectou infecção ativa e em 3 pacientes nos quais a "sPCR" detectou (coeficiente Kappa 0,57, acordo moderado). De 14 pacientes sintomáticos, 2 pacientes soronegativos para o HCMV que receberam rins soropositivos desenvolveram infecção primária. Analizando os dois métodos para o diagnóstico da infecção ativa, nós observamos maior sensibilidade e valor preditivo negativo da "L-PCR" ("L-PCR" 100% vs. "sPCR" 62%), e maior especificidade e valor preditivo positivo da "sPCR" ("sPCR" 81% vs. "L-PCR" 50% e 72%). O valor dos testes positivos, "L-PCR" e "sPCR", para predizer doença por HCMV foram, respectivamente, 57% e 70%, e o valor dos testes negativos para predizer que doença não desenvolveria foi 88% para "L-PCR" e 74% para a "sPCR". A análise comparativa entre os primeiros resultados positivos para a presença de infecção ativa por HCMV e o aparecimento de sintomas mostrou que a "L-PCR" precedeu o inicio dos sintomas clínicos 19 dias (mediana) em 8 pacientes. Em 2 pacientes, a "sPCR" precedeu 7 dias (mediana) a "L-PCR" na detecção de infecção sintomática por HCMV. A "L-PCR" e a "sPCR" foram considerados métodos complementares para o diagnóstico e monitoramento da infecção sintomática por HCMV. / Abstract: The Human Cytomegalovirus (HCMV) is the main cause of post-transplant complications. Specific methods that allow to identify early patients with risk of developing disease for which treatment is indicated, have been required so that few patients are treated unnecessarily and for the effective therapeutic institution. The "Nested-PCR" has been commonly used in the diagnosis of HCMV infection, however, when performed in peripheral blood leukocytes, due to its high sensitivity does not present good correlation with the development of HCMV disease. Detection of HCMV DNA in serum by PCR has been associated with the development of HCMV disease. In this study, we apply the "Nested-PCR" in peripheral blood leukocytes (termed "L-PCR"), the conventional method of Laboratory, and in serum ("sPCR"), for the diagnosis of active HCMV infection, in order to establish the correlation of results obtained of both methods with the development of symptomatic infection. With this purpose, we evaluated prospectively samples of 37 patients, 20 undergoing kidney transplantation, and 17 submitted to haematopoietic stem cells transplantation. To exclude false negative results in the amplification reaction by "sPCR", an internal control was constructed and all reactions were carried out by using it. According to the criteria established in this study, 21 patients (21/37 - 57%) developed active HCMV infection. All patients with active HCMV infection were positive by LPCR (p= 0,0003). The serum PCR were active HCMV infection positive for only 10 patients (10/37 - 27%) (p= 0,01). Discordant results were observed in 11 patients who had active HCMV infection positive for "L-PCR" but negative for "sPCR", of which 5 developed clinical symptoms for HCMV. The observed Kappa coefficient of agreement for both assays was 0,44 (moderate agreement). Sixteen of 37 patients (43%) did not develop active HCMV infection by neither of the methods employed, however 2 of these developed clinical symptoms of probable HCMV disease and one presented later confirmative biopsy for HCMV disease. Clinical symptoms were observed in 14 patients, in 12 of whom active infection was diagnosed by "LPCR" (p= 0,007) and in 7 by "sPCR" (p= 0,02). Absence of clinical symptoms were observed in 9 patients in which the "L-PCR" detected active HCMV infection and in 3 patients in which the "sPCR" detect it (kappa coefficient 0,57, moderate agreement). Of the fourteen symptomatic patients, 2 HCMV-seronegative patients who received seropositive Kidneys developed primary infection. Analyzing the two methods for the diagnosis of active infection, we observed higher sensitivity and negative predictive value of the "L-PCR" ("L-PCR" 100% vs. "sPCR" 62%), and higher specificity and positive predictive value of "sPCR" ("sPCR" 81% vs. "L-PCR" 50% e 72%). The value of positive "L-PCR" or "sPCR" tests to predict HCMV disease were respectively 57% and 70% and, the value of the negative tests to predict that HCMV disease would not develop was 88% to "L-PCR" and 74% to "sPCR". The comparative analysis between the first positive results for the presence of active HCMV infection and the onset of symptoms, showed that the "L-PCR" test preceded the onset of clinical symptoms 19 days (median) in 8 patients. In two patients the "sPCR" preceded 7 days (median) the "L-PCR" in the detection of symptomatic HCMV infection. Therefore the "L-PCR" and the "sPCR" were considered complementary methods for the diagnosis and manegement of HCMV symptomatic infection. / Mestrado / Mestre em Farmacologia

L'infection par le cytomégalovirus humain (HCMV) est une cause majeure de mortalité chez les patients immunodéprimés et représente la première cause d'infection congénitale. HCMV est un virus complexe qui s'est adapté au système immunitaire humain en développant de multiples mécanismes d'évasion. L'infection primaire à HCMV est associée à une réplication virale prolongée avant l'établissement de la latence. Il a été montré que cette intense réplication lors de la phase initiale de l'infection était associée à une épuisement fonctionnel des lymphocytes T CD4 spécifiques du virus. Alors que les anticorps jouent un rôle dans la limitation de la dissémination virale et la prévention de l'infection à HCMV, les réponses des lymphocytes B sont peu caractérisées. Dans le présent travail, nous avons étudié l'impact de l'infection à HCMV sur le phénotype et la fonctionnalité des sous-populations de LB du sang circulant chez une cohorte de femme enceintes avec une primo-infection par HCMV en utilisant comme contrôles des sujets sains séropositifs et séronégatifs pour HCMV ainsi que des femmes enceintes séronégatives. Nous montrons que l'infection primaire par HCMV induit une expansion significative et prolongée de deux sous-populations de LB :les LB mémoires activés (CD27+CD21low) et mémoires atypiques (CD27-CD21low), précédemment décrites lors d'infection chroniques. Les LB mémoires atypiques démontrent des signes d'épuisement fonctionnel comme en témoigne une expression élevée de récepteurs inhibant le BCR et une moindre réponse à la stimulation in vitro mesurée par la production de TNF-α. Les expansions de ces deux sous-populations sont corrélées entre elles et liées à la virémie. Ces résultats contribuent à la compréhension de la régulation des réponses des LB lors d'infections virales, en montrant que l'épuisement fonctionnel de LB, précédemment décrit lors d'infections chroniques, peut également survenir lors d'infections primaires.<p>Dans un deuxième temps, nous avons étudié l'acquisition des réponses B mémoires spécifiques de HCMV dirigées contre la principale glycoprotéine de surface, la glycoprotéine B (gB), et deux polypeptides du tégument. Lors de l'infection primaire par HCMV, la production d'anticorps neutralisant le virus, dirigés contre les glycoprotéines d'enveloppe, est retardée par rapport aux anticorps dirigés contre le tégument qui sont non neutralisant. Nous montrons que le phénotype des LB mémoires spécifiques de gB est différent de celui des LB mémoires spécifiques du tégument. La majorité des LB mémoires spécifiques de gB exprime un phénotype CD27+CD21+ alors que la majorité de ceux du tégument exprime le phénotype CD27+CD21low. Nous montrons par la suite chez des sujets sains que ces deux sous-populations de LB mémoires présentent des différences phénotypiques, au niveau de l'expression de récepteurs liés au "trafficking" cellulaire ainsi qu'au niveau de la fonctionnalité. Les LB mémoires CD21low, contrairement au LB mémoires CD21high, expriment des taux bas des récepteurs CXCR5 et CCR7, qui permettent la migration vers les centres germinatifs, mais des taux élevés de CD11c promouvant la migration vers les tissus périphériques. Après stimulation in vitro, les LB mémoires CD21low vont avoir une capacité de production d'immunoglobulines immédiate mais une réponse proliférative plus faible comparée aux LB mémoires CD21+. Nous démontrons la relevance de cette division des LB mémoires sur base de l'expression du CD21 dans un modèle de vaccination de rappel contre la toxoïde tétanique (TT). Après rappel, nous observons une expansion significative de LB mémoires spécifiques de la TT exprimant un phénotype CD27+CD21lowCXCR5lowCD11chigh. Nous proposons ainsi un nouveau mécanisme de manipulation des réponses humorales par des pathogènes qui se traduit par une limitation de l'induction de réponses B effectrices. Nos travaux permettraient également une meilleure approche des réponses B mémoires physiologiques chez l'homme en proposant une classification des LB mémoires basées sur leur fonctionnalité et leur phénotype.<p><p>Human cytomegalovirus (HCMV) infection is a major cause of mortality in immunocompromised patients and is the first cause of congenital infection worldwide. HCMV is a complex virus that has developed multiples immune evasions mechanisms during its co-evolution with mankind. Although often asymptomatic, primary HCMV infection is associated with an intense and prolonged viral replication. It has been previously shown that this intense viral replication is associated with functional exhaustion of virus-specific CD4+ T cells. Although neutralizing antibodies limits viral dissemination and play a role in the prevention of HCMV infection, B cell responses during HCMV infection have been poorly studied so far.<p>In this work, we have studied the impact of HCMV infection on the phenotype and functionality of peripheral-blood B cell subsets in a cohort of pregnant women with a primary HCMV infection. Controls were healthy seronegative and seropositive HCMV donors and HCMV seronegative pregnant women. We show that primary HCMV infection induces a significant and prolonged expansion of two B-cell subsets, previously described in chronic infections :activated memory B cells (MBC) (CD27+CD21low) and atypical MBC (CD27-CD21low). Atypical MBC display signs of functional exhaustion with increased expression of inhibitory receptors and a lower response to in vitro stimulation as assessed by TNF-α production. Expansion of these two subsets are correlated and higher in subjects with detectable viremia. These results contribute to the understanding of the regulation of B cell responses during viral infections and indicate that B cell exhaustion, previously described during chronic infections, can be observed in primary infection.<p>Next, we have characterized the acquisition of HCMV-specific B cell responses directed against envelope glycoprotein B (gB) and two tegument polypeptides (pp150 and pp52). During primary HCMV infection, the production of neutralizing antibodies targeting envelope glycoproteins is delayed when compared to non-neutralizing anti-tegument antibodies. We show that gB and tegument-specific MBC have distinct phenotype during primary HCMV infection. The majority of gB-specific MBC have a CD27+CD21+ phenotype while the majority of tegument-specific MBC have a CD27+CD21low phenotype. We show that CD27+CD21+ and CD27+CD21low MBC express different pattern of chemokine receptors pattern but also have distinct functionality. CD27+CD21low MBC, on the contrary to CD27+CD21+ MBC, express low levels of CXCR5 and CCR7 that favor migration to lymph nodes and germinal centers but express high levels of CD11c that promotes migration to inflammatory tissues.<p>In vitro stimulation of sorted subsets of healthy individuals indicates that CD27+CD21low MBC have higher capacity of immediate immunoglobulin production but a lower proliferative potential as compared to CD27+CD21+ MBC. We further show the relevance of a division of MBC subsets based on CD21 expression in a model of TT booster immunization. Following booster immunization, a significant expansion of TT-specific MBC expressing the phenotype CD27+CD21lowCXCR5lowCD11chigh is observed. <p>We propose that HCMV manipulates the host humoral response by limiting the induction of gB-specific CD27+CD21low "effector" MBC. Our work also indicates that human MBC physiological responses should be studied according to their respective phenotype and functions.<p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Contexte L'infection congénitale à cytomégalovirus (CMV) est la principale cause non génétique de déficience neurosensorielle. Bien que largement méconnues du grand public, ses manifestations néonatales ont un impact plus important que celui de nombreuses autres maladies pour lesquelles un dépistage prénatal est proposé. Actuellement, le dépistage prénatal de l'infection congénitale à CMV au Québec est basé sur l’échographie morphologique au deuxième trimestre. Cependant, l’échographie prénatale n'est ni un outil de dépistage sensible ni spécifique. Le dépistage sérologique est également possible : il permet d'évaluer le risque d'infection congénitale à CMV secondaire à une infection primaire à CMV, et de proposer une prophylaxie de la transmission verticale par antiviraux. Objectif Ce projet vise à comparer trois stratégies de dépistage pour évaluer si le dépistage sérologique universel de l'infection primaire à CMV pendant la grossesse est plus efficient en termes de ratio coût-utilité que le dépistage échographique prénatal seul ou le dépistage sérologique ciblé des femmes à haut risque. Méthodes Un modèle d'analyse décisionnelle et d'efficience a été élaboré, évaluant trois stratégies de dépistage pour une cohorte hypothétique de 80 000 naissances au Québec : le dépistage sérologique universel, le dépistage des femmes à haut risque uniquement, et le dépistage basé sur l'échographie prénatale seule. Les probabilités et les coûts ont été tirés respectivement de la littérature et des données nationales québécoises. Le principal résultat est exprimé en années de vie ajustées sur la qualité (QALY), avec un seuil de coût de 50 000 CAD par QALY perdu évité. Trois patients partenaires ont contribué à la conception de l'étude afin de cibler les critères d'utilité les plus pertinents. Résultats Dans le cadre de notre analyse comparative des différentes approches de dépistage, le dépistage universel s'avère plus efficace que le dépistage échographique et le dépistage ciblé sur les femmes à haut risque dans 95 % des cas. Dans les 5 % restants, le dépistage ciblé sur les femmes à haut risque est la meilleure stratégie de dépistage. Cette conclusion est également étayée par des analyses de sensibilité adaptées à diverses situations, y compris en l'absence d'efficacité du valacyclovir pour prévenir la transmission verticale. La mise en place de ce dépistage permettrait de prévenir 29 cas d'infection congénitale à CMV par an, avec un coût supplémentaire de 96 037,76 CAD par cas évité. Conclusion Le dépistage sérologique universel de l’infection primaire à CMV présente un rapport coût-utilité plus avantageux que la stratégie de dépistage standard au Québec, basée sur l'échographie morphologique. / Background Congenital Cytomegalovirus (CMV) infection is the primary cause of non-genetic sensorineural impairment in childhood. Although largely unknown to the public, its neonatal manifestations have a greater impact than many other diseases, for which prenatal screening is offered. Currently, prenatal screening for congenital CMV infection in Quebec is based on morphological ultrasound in the second trimester. However, prenatal ultrasound is neither a sensitive nor a specific screening tool. Serological screening is also possible: it allows for assessing the risk of congenital CMV infection following a primary CMV infection and to offer prophylaxis of vertical transmission with antivirals. Objective This project aims to compare three screening strategies to assess whether universal serological screening for primary CMV infection during pregnancy has a better cost-utility ratio than prenatal ultrasound screening alone or targeted screening of high-risk women. Study design A decision analysis and cost-effectiveness model was developed, evaluating three screening strategies for a hypothetical cohort of 80,000 births in Quebec: universal serological screening, screening of high-risk women only, and screening based on prenatal ultrasound alone. Probabilities and costs were derived from the literature and Quebec national data, respectively. The primary outcome is expressed in quality-adjusted life years (QALYs), with a cost threshold of 50,000 CAD per QALY lost avoided. Three patient partners contributed to the study design to target the most relevant utility criteria. Results In our comparative analysis of different screening approaches, universal screening demonstrates a superior cost-utility ratio in 95% of cases compared to ultrasound-based screening and screening of high-risk women only. In the remaining 5% of cases, targeted screening for high-risk women proves to be the optimal strategy. This conclusion is supported by sensitivity analyses tailored to various scenarios, including instances where valacyclovir lacks efficacy in preventing vertical transmission. Implementation of this screening could prevent 29 cases of congenital CMV infection per year, with an additional cost of 96,037.76 CAD per case avoided. Conclusion Our research suggests that a population-based maternal serological screening exhibits a more favorable cost-utility ratio for serological CMV screening than Quebec's current standard procedure, which is based on the ultrasound examination during pregnancy.

Neutralisierende Antikörper sind entscheidend in der Eindämmung der Virusinfektion, indem sie den Eintritt in die Wirtszelle hemmen bzw. die Aktivierung der Komplementkaskade initiieren. Distinkte wirtseigene Oberflächenrezeptoren für die Fc-Domäne von IgG (FcyR) sind für die Kommunikation von humoraler und zellulärer Immunantwort verantwortlich. Auch Mitglieder der Herpesviren kodieren für Fc-bindende Proteine, die Kandidaten für immunevasive Funktionen darstellen könnten. Der Nachweis der HCMV-kodierten Fc-bindenden Proteine gp34 und gp68 als Bestandteil der Virushülle lies auf eine immunevasive Funktion hinsichtlich neutralisierendem IgG und Komplement-vermittelter Virolyse schließen, wie für den HSV-1-kodierten FcyR gE beschrieben. Weder für gp34 noch für gp68 konnte in vitro ein hemmender Effekt auf Neutralisation und Virolyse beobachtet werden. In unserem Labor wurde jedoch gezeigt, dass gp34 und gp68 selektiv die IgG-abhängige Aktivierung zellulärer FcyR inhibieren. Die glykosylierungsunabhängige Ligandenbindung von gp34 und gp68 wies auf unterschiedliche Interaktionsmechanismen zwischen den zellulären und den viralen FcyR hin. Mithilfe eines mutierten Fc-Fragments konnte für gp68 eindeutig eine mit HSV-1 gE überlappende Bindestelle an IgG identifiziert werden. Die für die Ligandenbindung erforderlichen Aminosäuren 71-292 von gp68 binden Fc in einer 2:1 Stöchiometrie, wobei die N-, nicht aber die O-Glykosylierung des vFcyRs essentiell sind. Darüber hinaus formt gp34 auf infizierten Zellen und auf der Virushülle kovalente Homooligomere. gp34-Cysteinpunktmutanten auf Basis der für die Bindung notwendigen Aminosäuren 24-140 lassen vermuten, dass die Oligomerisierung Voraussetzung für die Fc-Bindung ist. Im Gegensatz zu gp68 scheint der Mechanismus der Fc-Bindung von gp34 einzigartig unter den bekannten Fcy-Rezeptoren zu sein. Diese Ergebnisse lassen vermuten, dass trotz redundanter Expression der HCMV-FcyR der Bindungs- und Wirkungsmechanismus selektiv ist. / Neutralizing IgGs play a key role in diminishing virus infectivity by inhibiting the entry into host cells. Additionally, IgG-bound particles may be inactivated by virolysis through the activation of complement. Surface receptors specific for the Fc domain of IgG represent host proteins, connecting humoral and cellular immune responses. Also members of the herpes virus family code for proteins with Fc binding properties, implying functions that could intervene with antibody-dependent effector mechanisms. The presence of gp34 and gp68 on the virion membrane raised the question whether they are able to inhibit neutralising IgG and complement-mediated virolysis. The HSV-1-encoded FcyR gE was described to affect neutralisation and virolysis. Despite extensive analysis, there were no implications found that gp34 or gp68 interfere with neutralising IgG or virolysis in vitro. However, our lab could demonstrate that gp34 and gp68 selectively inhibit the IgG-dependent activation of the different host FcyRs. In contrast to the cFcyRs Fc recognition by gp34 and gp68 occurs independently of N-linked glycosylation of IgG, which points to a different binding mechanism among host and viral FcyRs. By taking advantage of a mutated Fc fragment, overlapping binding regions of the HSV-1 gE and gp68 were identified. For gp68 the amino acids 71-292 including the N-glycans are strictly required for Fc binding in a 2:1 stoichiometry. Interestingly, gp34 forms covalently linked homo-oligomers in infected cells and on the virion. Based on the minimal binding domain comprising the amino acids 24-140 of gp34, targeted cysteine exchange mutants revealed that oligomer formation by gp34 is absolutely required for Fc binding. In contrast to gp68, the Fc binding characteristics of gp34 appears to be unique among the known FcyRs. These findings allow us to postulate that even if the HCMV-encoded FcyRs are redundantly expressed the mechanistic details and binding properties are selective.

Die Mehrzahl der im Thymus generierten CD4+CD25high regulatorischen T-Zellen (Treg) besitzt hohe Affinität gegenüber körpereigenen Antigenen. Es ist bekannt, dass T-Zell Rezeptoren (TCR) auf Treg Zellen in der Peripherie zusätzlich auch fremde Antigene verschiedener Pathogene wie Parasiten, Bakterien und Viren erkennen. Wenig ist bekannt über das klonale T-Zell Rezeptor Repertoire dieser Treg Populationen und ihre Beziehung zu CD4+CD25low effektor T-Zellen (Teff) im Menschen. In dieser Studie analysieren wir humane TCR auf expandierten Treg and Teff Zellen mit definierter Antigen Spezifität für Haupthistokompatibilitätskomplex (MHC) Klasse II restringierte „fremde“ Epitope des Cytomegalovirus (CMV). Bemerkenswerterweise fanden wir, dass der gleiche TCR Vb-CDR3 Klon in beiden funktionell unterschiedlichen Subpopulationen in vitro dominant expandiert ist. Im Unterschied zu ihren klonal-identischen Teff Gegenspielern, exprimieren die suppressiven Treg Zellen kaum CD127 und IL-2, aber hohe Mengen an IFNg und IL-10. Zusammen mit der signifikant erhöhten FOXP3 Expression, trotz unvollständiger foxp3-DNA Demethylierung, lassen sich die CMV-spezifischen CD4+CD25high Treg Zellen einem induzierten Treg (iTreg) Phänotyp zuordnen mit Ähnlichkeit zum beschriebenen Tr-1 Phänotyp. Darüber hinaus konnten wir die klonale TCR Identität auch in frisch isolierten CD4+CD25low und CD4+CD25high Subpopulationen bestätigen, was die Entstehung von CMV-spezifischen Treg Zellen bereits in vivo nahe legt. Periphere CD25high Treg Zellen supprimieren die anti-virale Immunantwort in Patienten mit häufigen CMV-Reaktivierungen, was auf ihre Bildung als Reaktion chronischer Antigenexposition interpretiert werden kann. Unsere Ergebnisse beweisen erstmals direkt, dass aus dem gleichen humanen T-Zell Klon Teff und Treg Zellen mit identischer Spezifität entstehen können und lassen vermuten, dass die Treg Induktion in der Peripherie durch häufige Antigenexposition vorangetrieben wird. / The majority of thymically arised regulatory CD4+CD25high T cells (Treg) show high affinity to self-antigens. It has been proposed that T-cell receptors (TCR) on Treg cells in the periphery also recognize foreign-antigens from pathogens, such as bacteria and viruses. Studies in mice have shown that peripheral Treg cells can be generated not only from naïve T cells but also from effector T cells (Teff). However, in humans the clonal TCR-repertoire of these Treg populations and their relation to effector CD4+CD25low Teff is not sufficiently known up to date. Here, we analyzed human TCRs derived from expanded Treg and Teff cells with defined specificity to MHC class-II restricted “foreign” epitopes of Cytomegalovirus (CMV). Remarkably, we found that both functionally distinct subsets share the same dominant TCR-CDR3 clones in vitro. In contrast to their Teff counterparts, the Treg cells express low CD127 and IL-2, but high IL-10 upon antigen stimulation. Therefore, together with increased FOXP3 expression, but incomplete foxp3 DNA-demethylation, human CMV-antigen specific Treg cells exhibit an induced phenotype (iTreg) in vitro with similarity to recently described Tr-1 phenotype. Moreover, the clonal identity was confirmed in freshly isolated CD4+CD25low and CD4+CD25high subsets, suggesting their generation occurred already in vivo. Peripheral CD25high Treg cells suppress the anti-viral immune response in patients with frequent CMV-reactivations, implying their development as reaction on chronic antigen-exposure. Our results demonstrate directly for the first time, that the same human T-cell clone can possess the phenotype of Teff and Treg cells with specificity to identical foreign epitopes and suggest that Treg-induction in the periphery is supported by frequent antigen-exposure.

The murine cytomegalovirus (MCMV) immediate early 1 (IE1) protein has been described as a trans-activator of viral and host gene expression. However, the precise role that IE1 plays in the viral life cycle, and in particular its effect on the host immune response is not known. This thesis investigates the functional relationship of the IE1 protein and the immune response induced after infection. By using an ie1-deletion mutant MCMV (MCMVdie1) it was demonstrated that, early after infection, tumor necrosis factor (tnf ) gene activation and protein production was significantly induced in infected-primary macrophages (M ) to a much greater extent than its wild type counterpart. In addition, preliminary studies on the signalling pathways activated upon infection were carried out in order to gain information about the pathways that might be involved in MCMVinduced modulation of tnf activation. Initial observations on the MAPK family members Erk1/2, p38 and JNK did not revealed any differential activation in the absence of IE1. However, due to a number of limitations, it was not possible to draw any firm conclusions from this study. Investigation of the role of IE1 in the in vivo production of TNF were also performed in both susceptible (BALB/c) and resistant (C57Bl/6) mice. These experiments confirmed the attenuated phenotype of MCMVdie1 in vivo, whereby the mutant strain grew to much lower titers than wild type. When cytokine production was assessed in relation to PFU levels a significant production of TNF after infection is observed in different organs of both mice strains. This raises the question whether IE1 contributes to MCMV modulation of TNF production in the natural host. Although, because it is still unclear whether the phenotype of MCMVdie1 in vivo is due to a defect in the virus or the result of a immune response, it was not possible to conclude unequivocally that IE1 is responsible for dampening this cytokine response. This thesis also tested whether the attenuated replication of MCMVdie1 in vivo was due to the increased TNF production induced after infection. An initial investigation in tnf depleted mice revealed that the MCMVdie1 growth phenotype is not due to TNF response. Overall, this study has provided insight into a potential immune modulatory function by MCMV associated with IE1 protein and the regulation of TNF in vivo and in vitro.

Aim: To analyze differences in patients’ clinical course, we compared two regimes of either preemptive therapy or prophylaxis after liver transplantation. Methods: This retrospective study was reviewed and approved by the institutional review board of the University of Leipzig. Cytomegalovirus (CMV) prophylaxis with valganciclovir hydrochloride for liver transplant recipients was replaced by a preemptive strategy in October 2009. We retrospectively compared liver transplant recipients 2 years before and after October 2009. During the first period, all patients received valganciclovir daily. During the second period all patients included in the analysis were treated following a preemptive strategy. Outcomes included one year survival and therapeutic intervention due to CMV viremia or infection. Results: Between 2007 and 2010 n = 226 patients underwent liver transplantation in our center. n = 55 patients were D+/R- high risk recipients and were excluded from further analysis. A further 43 patients had to be excluded since CMV prophylaxis/preemptive strategy was not followed although there was no clinical reason for the deviation. Of the remaining 128 patients whose data were analyzed, 60 received prophylaxis and 68 were treated following a preemptive strategy. The difference in overall mortality was not significant, nor was it significant for one-year mortality where it was 10% (95%CI: 8%-28%, P = 0.31) higher for the preemptive group. No significant differences in blood count abnormalities or the incidence of sepsis and infections were observed other than CMV. In total, 19 patients (14.7%) received ganciclovir due to CMV viremia and/or infections. Patients who were treated according to the preemptive algorithm had a significantly higher rate risk of therapeutic intervention with ganciclovir [n = 16 (23.5%) vs n = 3 (4.9%), P = 0.003)].

Firstly, the aims of the study were to investigate the influences of various factors, sex differences, Cytomegalovirus/Epstein-Barr virus (CMV/EBV) serostatus and vitamin D concentrations on respiratory illness incidence and immune function during the winter months in a student cohort of endurance athletes. In Chapter 3, the findings of the study concur with recent reports of illness incidence at major competitive games which indicate that female athletes may be more susceptible than their male counterparts to upper respiratory tract illness (URTI) symptoms and that lower oral-respiratory mucosal immunity may, in part, account for this. It was also found that previous coinfection with CMV and EBV might promote protective immune surveillance to lower the risk of URTI. In addition, it can be concluded that athletes with low plasma vitamin D concentrations may have a higher risk of URTI and suffer more severe symptoms when URTI is present. This may be due to impaired mucosal and systemic immunity as secretory immunoglobulin A (SIgA) secretion, cathelicidin levels and antigen-stimulated pro-inflammatory cytokine production appear to be increased by vitamin D-dependent mechanisms. A series of follow-up studies were also conducted to examine the effect of vitamin D on mucosal and systemic immunity in athletes. In Chapter 4, it was reported that the influence of vitamin D on circulating cytokines might be different in athletes compared with non-athletes and that both pro-inflammatory and anti-inflammatory cytokine production by multi-antigen stimulated whole blood culture were not influenced by 1,25-dihydroxy vitamin D (1, 25(OH)2D) iconcentrations within the normal healthy range. In Chapter 5, it was found that 5000 IU of vitamin D3 supplementation daily appears to have a beneficial effect in up-regulating the expression of SIgA and cathelicidin in athletes during a winter training period. Nevertheless, the findings reported in Chapter 6 showed that there were no significant effects of vitamin D status and a 4-week period of daily high does vitamin D3 supplementation on salivary antimicrobial protein (AMP) responses to prolonged exercise. In conclusion, a series of studies in this thesis have demonstrated the influence of various factors (sex differences, CMV/EBV serostatus and vitamin D concentrations) on susceptibility to URTI among athletes. Moreover, it was suggested that vitamin D3 supplementation could have a positive effect on immune function and lead to decreased incidence of respiratory infections.