Thesis (Ph.D. in Epidemiology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 114-126).

La macroautophagie assure l'homéostasie cellulaire en recyclant du matériel cytosolique obsolète ou délétère et sa dérégulation est associée à plusieurs pathologies. Elle constitue aussi un mécanisme de défense car elle peut éliminer des pathogènes intracellulaires. L'étape cruciale de l'autophagie est la maturation lors de laquelle la vésicule renfermant des substrats cytosoliques, l'autophagosome, fusionne avec des lysosomes et la dégradation a lieu. Nous nous intéressons à la régulation de l'autophagie et aux conséquences de sa perturbation lors des infections, notamment par le virus de la rougeole (VR). Les données de l'équipe montrent qu'il induit et utilise toutes les étapes de l'autophagie, afin de se répliquer efficacement. Mes travaux montrent que des protéines du virus peuvent interagir avec au moins deux protéines cellulaires NDP52 et T6BP qui sont des récepteurs autophagiques (protéines cytosoliques ayant un domaine de liaison aux autophagosomes et un domaine de liaison au substrat à dégrader, par exemple des pathogènes). J'ai alors étudié le rôle des récepteurs autophagiques T6BP, NDP52 et Optineurine dans la réplication virale. J'ai aussi participé à une étude décrivant que NDP52 et Optineurine régulent en plus la maturation. Mes travaux de thèse démontrent un tel double rôle pour T6BP. Cependant, seuls T6BP et NDP52 sont nécessaires à la réplication du VR bien qu'elle requiert la maturation autophagique. Ainsi mes résultats suggèrent d'une part que les trois récepteurs puissent réguler la maturation d'autophagosomes distincts.D'autre part, le VR pourrait exploiter individuellement les autophagosomes dont la maturation dépend de T6BP et NDP52 pour se répliquer / Macroautophagy ensures cell homeostasis through the recycling of obsolete or deleterious cytosolic components and its deregulation is associated with several pathologies. It is also a defense mechanism as it allows the elimination of intracellular pathogens. The most important autophagic step is maturation, during which the cytosolic substrate-containing vesicle, the autophagosome, fuses with lysosomes and the degradation occurs. We study autophagy regulation and the consequences of its disruption during infections and in particular by measles virus (MeV). Our team has shown that MeV induces and exploits all steps of autophagy, to replicate more efficiently. My results indicate that viral proteins can interact with at least two cellular proteins, NDP52 and T6BP, which are autophagy receptors (cytosolic proteins that carry an autophagosome-binding domain and a domain binding substrates that would be degraded, such as intracellular pathogens). I then studied the role of autophagic receptors T6BP, NDP52 and OPTINEURIN in viral replication. I also took part in a study describing NDP52 and OPTINEURIN as autophagosome maturation regulators. My work depicts the same dual role for T6BP. However, only T6BP and NDP52 are necessary for MeV replication even though it requires autophagosome maturation. Thus, my results suggest that the three autophagy receptors might regulate distinct autophagosome maturation on one hand. On the other, MeV could individually exploit autophagosomes, the maturation of which is regulated by T6BP or NDP2 to replicate efficiently

Inledning: Trots ansträngningar från nationella och internationella myndigheter har följsamheten till det rekommenderade vaccinet mot mässling minskat. Detta har resulterat i ett ökat antal rapporterade fall av mässling i världen, vilket riskerar ha allvarliga konsekvenser för befolkningen. Syfte: Studiens syfte är att undersöka och sammanställa den vetenskapliga litteraturen om vilka faktorer som ligger bakom föräldrars beslut gällande att inte låta sina barn vaccineras mot mässling. Metod: Litteraturstudie med deskriptiv design. Systematiska sökningar genomfördes i databaserna Academic Search Elite, Cinahl & PubMed. Data analyserades och kategoriserades under fem teman. Resultat: De flesta av föräldrarna var ovetandes om riskerna med att drabbas av mässling och oroades istället över konsekvenserna av eventuella bieffekter till följd av vaccinet. Deras attityd till vaccinet var även starkt påverkad av deras individuella tro och livsstil, likväl deras sociala omgivning. Föräldrarna rapporterade även brist i tillförlitlig information samt en oförmåga att fatta informerade beslut. Diskussion: Bristen i överensstämmelse med aktuella riktlinjer för vaccination av små barn mot mässling är ett multifaktoriellt samhällsproblem och som varierar mellan olika befolkningsgrupper. Föreliggande studier understryker behovet av effektivare kommunikationsstrategier för att hjälpa föräldrar att förstå fördelarna med barnvaccination. / Introduction: Despite intense efforts made by national and international health authorities to promote the importance of measles vaccination, the prevalence of vaccinated children has been decreased.Aim: The aim of this study was to perform a systematic review of the scientific literature to describe the factors supporting parental decision to refuse childhood measles vaccination.Method: A systematic review of qualitative studies was conducted in the following databases: Academic Search Elite, Cinahl and PubMed. Data was analyzed and the results were classified into five categories. Result: Most parents were unaware of the risks connected to measles and were instead more concerned about the consequences of potential side effects of the vaccine. The parents’ attitude to vaccination was strongly influenced by their individual beliefs and lifestyle, as well as by the interaction with the social environment. The parents also reported a lack of reliable information and the consequent inability to take an informed decision.Discussion: The lack of compliance to the guidelines for the vaccination of young children against measles is influenced by factors varying between different population groups. Notably, this study highlighted the need for more effective communication strategies to help parents understand the advantages connected to children’s vaccination.

This thesis focuses on the epidemiological situation in the incidence of measles in the Czech Republic and possibilities of influencing their unfavourable situation in Europe. Measles is a highly contagious disease of viral origin, which has a significant impact on the health of the child population in particular. The most effective protection against the measles is vaccination. The introduction of measles vaccination positively affected the epidemiological situation in the world. Priority of the WHO (World Health Organization, WHO, hereinafter) is elimination of the measles. The long term goal of the WHO and the European Centre for Disease Prevention and Control (ECDC hereinafter) was the elimination of measles in the European region by 2010. This goal was not achieved. The cause was low immunization coverage of certain population groups in several European countries. The prerequisite of achieving elimination of measles is that the vaccination coverage in the population is high. Many countries, thanks to effective vaccination strategy, succeeded in reducing the incidence of measles. In the years 2007- 2009 the historically lowest incidence, of less than 10 measles cases per million inhabitants, in the European region was achieved. In subsequent years, there was again a rise in measles morbidity. A repeated rise in cases of the disease is attributed to the expansion of social groups where vaccination coverage was inadequate in terms of achieving herd immunity. The thesis is divided into theoretical and practical part. The theoretical part is divided into four main chapters. The first chapter describes the history of measles, the origin of the name of the disease and the first mention of it. The second theoretical chapter is devoted to the clinical characteristics of measles. In subchapters it describes the clinical picture of the disease, possible complications of the disease, how the infection is diagnosed and what treatment options there are. The third chapter defines the epidemiological characteristics of the disease. The subheads are aimed at the cause of the disease, possible ways of transmission, incubation period, period of communicability, susceptibility and incidence of this disease. At last, the fourth chapter of the theoretical part desribes possible epidemiological measures, which include measles surveillance, preventive and repressive measures and vaccination. The practical part of the thesis was elaborated through quantitative research - secondary data analysis. Analysed data were obtained from the information system for reporting and recording infectious diseases EPIDAT on Regional Hygiene Station of South Bohemian Region, based in Budweis and the European Centre for Disease Prevention and Control. The data on population, in terms of age groups and individual regions, were drawn from the demographic yearbooks, which are available on the website of the Czech Statistical Office. The resulting data were subsequently processed in tabular and graphical forms in Microsoft Excel. The research objectives were: 1) to set charting trends in the incidence of measles in the Czech Republic and Central Europe over the period 2004-2013, 2) to compare the incidence of measles in the individual regions of the Czech Republic, and 3) to identify the reasons for gaps in vaccination coverage against measles in the South Bohemian Region. Based on the above stated objectives, the five research questions were defined. The results are intended to highlight the issues of possible adverse influence on the epidemiological situation in the incidence of measles in Europe. This thesis can be used as a source of information on trends in incidence, vaccine development and immunization status for measles.

Le virus de la rougeole est un agent pathogène responsable d’immunosuppressions transitoires mais sévères chez les individus infectés. L’infection par ce virus peut ainsi mener à l’établissement d’infections secondaires opportunistes, souvent décrites chez les patients rougeoleux. Cependant, la contribution du virus de la rougeole sur des infections secondaires à l’échelle de la cellule co-infectée n’a jamais fait l’objet d’études. Notre équipe à précédemment démontré que le virus de la rougeole induit une autophagie productive dans les cellules infectées, requise pour une réplication optimale du virus. À l’opposé, certains pathogènes comme la bactérie Salmonella typhimurium sont restreints par l’autophagie. Le but de cette thèse est d’étudier la contribution de l’autophagie sur la prolifération bactérienne en condition de co-infection avec le virus de la rougeole. Au cours du projet, nous avons identifié que dans les cellules co-infectées avec le virus de la rougeole, la bactérie Salmonella typhimurium hyperprolifère. Cette prolifération intense prend place essentiellement dans des cellules multinucléées géantes (syncytia) formées par le virus. En outre, la bactérie, normalement localisée dans une vacuole cellulaire, se localise dans le cytosol de ces syncytia et semble insensible à l’autophagie. Au cours de cette thèse, nous avons identifié que le facteur antimicrobien TBK1 pourrait être détourné par l’infection virale, contribuant ainsi à l’échappement de la bactérie à l’autophagie. Ce travail de thèse met ainsi en évidence une nouvelle possibilité d’échappement de bactéries à l’autophagie lors d’une co-infection virale. / Measles virus is a pathogenic agent responsible for transient but severe immunosuppression in infected individuals. The infection can lead to the establishment of secondary infections, frequently described in measles virus infected patients. Nevertheless, Measles virus contribution to secondary infection at cell scale level have never been studied yet. Our team has previously described that Measles virus induce a fully functional autophagy in infected cells, which is mandatory for an efficient viral replication. On the opposite, some pathogens, as the bacteria Salmonella typhimurium are restricted by autophagy. The aim of this PhD project is to study the contribution of autophagy on bacterial proliferation upon Measles virus co-infection at cell level. During this project, we have identified that in Measles virus coinfected cells, Salmonella typhimurium hyperproliferates. This exacerbated proliferation takes place in multinucleated giant cells induced by the virus, which are called syncytia. In addition, the bacteria, which is normally localized in cellular vacuole, is localized directly inside the cytosol of syncytia. Furthermore, cytosolic bacteria appears to be insensitive to autophagy. During this PhD project, we have identified that the cellular factor TBK1 could be hijack by the viral infection. Thus, this could allow the auophagic escape of the bacteria. This study highlight a new opportunity of autophagic escape of bacteria during a viral co-infection.

abstract: Vaccines against the arthropod-borne dengue virus (DENV) are still commercially nonexistent. A subunit immunization strategy may be of value, especially if a safe viral vector acts as a biologically active adjuvant. The DENV envelope protein (E), the main target for neutralizing immune responses, has three conformational domains. The immunoglobulin-like and independently folding domain III (DIII) contains epitopes that elicit highly specific neutralizing antibodies. The hepatitis B small surface antigen (HBsAg, S) was used as a scaffold to display DENV 2 DIII on a virus-like particle (VLP). A measles virus (MV) was engineered to vector HBsAg and the hybrid glycoprotein DIII-HBsAg in two different loci (DIII-S). Despite the relatively deleterious effect on replication caused by the insertion of two transcription cassettes, the recombinant virus MVvac2(DIII-S,S)P induced the secretion of DIII-S hybrid VLP with a similar sucrose density as HBsAg particles (1.10-1.12g/ml) and peaked at 48 h post-infection producing 1.3x106 TCID50/ml infectious MV units in vitro. A second recombinant virus, MVvac2(DIII-S)N, was engineered to vector only the hybrid DIII-S. However, it did not induce the secretion of hybrid HBsAg particles in the supernatant of infected cells. The immunogenicity of the recombinant viruses was tested in a MV-susceptible small animal model, the experimental group which received two 105 TCID50 I.P. doses of MVvac2(DIII-S,S)P in a 28 day interval developed a robust immune response against MV (1:1280), HBsAg (787 mIU/ml) and DENV2 (Log10 neutralization index of 1.2) on average. In summary, it is possible to display DENV E DIII on hybrid HBsAg particles vectored by MV that elicit an immune response. This forms the basis for a potential vaccine platform against DENV. / Dissertation/Thesis / Masters Thesis Biology 2015

abstract: Despite the safe and effective use of attenuated vaccines for over fifty years, measles virus (MV) remains an insidious threat to global health. Problematically, infants less than one year of age, who are the most prone to severe infection and death by measles, cannot be immunized using current MV vaccines. For this dissertation, I generated and performed preclinical evaluation of two novel MV vaccine candidates. Based on data from clinical trials that showed increasing the dosage of current MV vaccines improved antibody responses in six-month-old recipients, I hypothesized that increasing the relevant antigenic stimulus of a standard titer dose would allow safe and effective immunization at a younger age. I generated two modified MVs with increased expression of the hemagglutinin (H) protein, the most important viral antigen for inducing protective neutralizing immunity, in the background of a current vaccine-equivalent. One virus, MVvac2-H2, expressed higher levels of full-length H, resulting in a three-fold increase in H incorporation into virions, while the second, MVvac2-Hsol, expressed and secreted truncated, soluble H protein to its extracellular environment. The alteration to the virion envelope of MVvac2-H2 conferred upon that virus a measurable resistance to in vitro neutralization. In initial screening in adult mouse models of vaccination, both modified MVs proved more immunogenic than their parental strain in outbred mice, while MVvac2-H2 additionally proved more immunogenic in the gold standard MV-susceptible mouse model. Remarkably, MVvac2-H2 better induced protective immunity in the presence of low levels of artificially introduced passive immunity that mimic the passive maternal immunity that currently limits vaccination of young infants, and that strongly inhibited responses to the current vaccine-equivalent. Finally, I developed a more physiological infant-like mouse model for MV vaccine testing, in which MV-susceptible dams vaccinated with the current vaccine-equivalent transfer passive immunity to their pups. This model will allow additional preclinical evaluation of the performance of MVvac2-H2 in pups of immune dams. Altogether, in this dissertation I identify a promising candidate, MVvac2-H2, for a next generation measles vaccine. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2016

abstract: Measles is a contagious, vaccine-preventable disease that continues to be the leading cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins. Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects. Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23). These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker. / Dissertation/Thesis / Masters Thesis Biology 2016

abstract: Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age, whom are particularly prone to Dengue severe infection and death, cannot be immunized using current approved DV vaccine. The most important issues documented so far are the lack of efficiency and enhancement of the disease in young seronegative recipients, as well as uneven protection against the four DV serotypes. Based on data from clinical trials that showed enhanced performance of dengue vaccines when the host has previous anti-flaviviral immunity, I proposed here an attractive solution to complement the current vaccine: a recombinant measles vaccine vectoring dengue protective antigens to be administered to young infants. I hypothesized that recombinant measles virus expressing Dengue 2 and 4 antigens would successfully induce neutralizing responses against DV2 and 4 and the vaccine cocktail of this recombinant measles can prime anti-flaviviral neutralizing immunity. For this dissertation, I generated and performed preclinical immune assessment for four novel Measles-Dengue (MV-DV) vaccine candidates. I generated four MVs expressing the pre membrane (prM) and full length or truncated (90%) forms of the major envelope (E) from DV2 and DV4. Two virus, MVvac2-DV2(prME)N and MVvac2-DV4(prME), expressed high levels of membrane associated full-length E, while the other two viruses, MVvac2-DV2(prMEsol)N and MVvac2-DV4(prMEsol)N, expressed and secreted truncated, soluble E protein to its extracellular environment. The last two vectored vaccines proved superior anti-dengue neutralizing responses comparing to its corresponding full length vectors. Remarkably, when MVvac2-DV2/4(prMEsol)N recombinant vaccines were combined, the vaccine cocktail was able to prime cross-neutralizing responses against DV 1 and the relatively distant 17D yellow fever virus attenuated strain. Thus, I identify a promising DV vaccination strategy, MVvac2-DV2/4(prMEsol)N, which can prime broad neutralizing immune responses by using only two of the four available DV serotypes. The current MV immunization scheme can be advantageus to prime broad anti-flaviviral neutralizing immunity status, which will be majorly boosted by subsequent chimeric Dengue vaccine approaches. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016

abstract: ABSTRACT In terms of prevalence, human suffering and costs dengue infections are the most important arthropod-borne viral disease worldwide. Dengue virus (DENV) is a mosquito-borne flavivirus and the etiological agent of dengue fever and dengue hemorrhagic fever. Thus, development of a safe and efficient vaccine constitutes an urgent necessity. Besides the traditional strategies aim at generating immunization options, the usage of viral vectors to deliver antigenic stimulus in order to elicit protection are particularly attractive for the endeavor of a dengue vaccine. The viral vector (MVvac2) is genetically equivalent to the currently used measles vaccine strain Moraten, which adds practicality to my approach. The goal of the present study was to generate a recombinant measles virus expressing structural antigens from two strains of DENV (DENV2 and DENV4) The recombinant vectors replication profile was comparable to that of the parental strain and expresses either membrane bound or soluble forms of DENV2 and DENV4 E glycoproteins. I discuss future experiments in order to demonstrate its immunogenicity in our measles-susceptible mouse model. / Dissertation/Thesis / M.S. Biology 2013