Le virus de la rougeole (MV) provoque une maladie sévère chez les enfants qui induit une forte immunosuppression et peut dans certains cas infecter le système nerveux central (SNC). La protéine CD150, principal récepteur cellulaire du virus, permet l’entrée du MV en se liant à l’hémagglutinine (MV-H). L’altération fonctionnelle des cellules dendritiques (DC) étant considérée comme essentielle dans l’immunopathogénèse du MV, nous avons analysé les conséquences de l’interaction de MV-H avec les DC. Nous avons développé un modèle expérimental qui nous permet d’étudier l’interaction directe entre CD150 et MV-H hors contexte infectieux. Nos résultats montrent que cette interaction provoque une diminution de l’expression des molécules de surface CD80, CD83, CD86, et HLA-DR, de la production d’IL-12 par les DC, et de la capacité des DC à stimuler la prolifération des lymphocytes T. L’interaction CD150-MV-H a inhibé la phosphorylation des protéines kinases JNK1/2 dans les DC et les lymphocytes B (LB) induite par la stimulation de CD150 par un anticorps spécifique, mais pas celle des kinases ERK1/2. Par ailleurs MV-H seule induit la phosphorylation d’Akt via CD150 dans les DC et les LB. La liaison de CD150 par MV-H a réduit la réponse inflammatoire chez les souris transgéniques exprimant CD150 humain, ce qui confirme l’effet de l’interaction de CD150 et MV-H in vivo. Nos études ont révélé l’expression de CD150 dans les tumeurs du SNC et l’existence d’une nouvelle isoforme de CD150. Cette isoforme contient un exon supplémentaire de 83 pb et est exprimée dans les cellules lymphoïdes et les DC en plus des tumeurs du SNC. Bien que l’expression de CD150 soit uniquement intracellulaire dans les cellules tumorales, elle peut représenter un nouveau marqueur pour les tumeurs cérébrales humaines. Cette étude apporte un éclairage nouveau sur le rôle immunorégulateur de CD150 et sur la diversité de ses isoformes, et ouvre ainsi de nouvelles perspectives pour leurs applications thérapeutiques. / Measles virus (MV) causes an acute childhood disease, associated in certain cases with the infection of the central nervous system (CNS). MV induces a profound immunosuppression, resulting in high infant mortality. The major cellular receptor for MV is CD150, which binds MV hemagglutinin (MV-H). As dendritic cell (DC) dysfunction is considered to be essential for the MV immunopathogenesis, we analyzed consequences of MV-H interaction with DCs. We developed an experimental model allowing us to analyze the direct CD150-MV-H interaction in the absence of infectious context. This interaction caused the downregulation of surface expression of CD80, CD83, CD86 and HLA-DR molecules and inhibition of IL-12 production in DCs. DCs also failed to activate T cell proliferation. The CD150-MV-H interaction in DCs and B cells decreased the phosphorylation of JNK1/2, but not ERK1/2 kinases, after subsequent CD150 ligation with anti-CD150 antibodies. Moreover, MV-H by itself induced Akt phosphorylation via CD150 in DCs and B cells. Engagement of CD150 by MV-H in mice transgenic for human CD150 decreased the inflammatory reaction, contact hypersensitivity response, confirming the immunosuppressive effect of CD150-MV-H interaction in vivo. Furthermore, our studies revealed the CD150 expression in CNS tumors and identified the novel CD150 isoform, containing an additional 83bp exon expressed in lymphoid cells, DCs and CNS tumors. Although its isoforms remain intracellular in tumor cells, CD150 may represent a new marker for human brain tumors. Novel mechanism of CD150-induced immunosuppression and new CD150 isoform identified in these studies shed new light on its immunoregulatory role and CD150 isoform diversity and open perspectives for their clinical applications.

Measles (MV) virions, like those of other enveloped viruses, enter cells by fusing their lipid membranes with those of the target host cells. Additionally, infected tissues often possess giant multinucleate cells, known as syncytia, which are formed by fusion of infected cells with uninfected neighbors. Expression of both the MV attachment (H) and fusion (F) proteins is required for membrane fusion. MV H mediates receptor binding in order to bring the two membranes into close proximity prior to F activation and is thought to trigger F activation through a specific interaction between the two proteins. Although measles H and F are efficiently transported to the cell surface when expressed independently, evidence has been reported in support of an intracellular interaction between the two proteins that can be detected using an ER co-retention approach. However, it was not determined if the putative co-retention was specific to the two measles glycoproteins, as is their ability to complement each other for efficient fusion promotion. Thus, in this thesis, the formation of an intracellular complex between MV H and F was re-examined. Consistent with the formation of an intracellular complex, cell surface expression and receptor binding of untagged wt MV H is slightly reduced by co-expression of an excess of ER-tagged MV F compared to co-expression with wt F. However, the reduction in surface expression is non-specific in that it can also be induced with heterologous proteins of NDV, which lack significant homology with those of MV. Although this approach did not detect a specific intracellular interaction between MV H and F, it cannot be ruled out that there is a weak association of the proteins that is undetectable by this method. This led to the use of an alternative approach to investigate the cellular site(s) of interaction between the measles H and F proteins. Consistent with a cell surface interaction between MV H and F, the combination of surface biotinylation and co-immunoprecipitation detects formation of a virus-specific H-F complex. Approximately, 21% of the total amount of MV H at the cell surface can be captured with MV F using an antibody against the latter protein. Two complementary approaches were used to address the relationship between this cell surface interaction and receptor recognition by MV H. First, the proteins were co-immunoprecipitated from the surface of Chinese hamster ovary (CHO) cells, which do not express either MV receptor, CD46 or CD150. Similar levels of MV H can be co-immunoprecipitated with F from the surfaces of parental CHO cells and stably transfected cells that express, human CD46 (CHO-CD46), indicating that binding to CD46 is not the trigger for the H-F interaction. Second, MV H proteins, carrying mutations that dramatically reduce CD46 binding, were shown to co-immunoprecipitate efficiently with F from the surface of HeLa cells. Significantly, these results indicate that MV H and F interact in the absence of, and thus prior to, receptor binding. This is in direct contrast to the NDV HN-F cell surface interaction, which is thought to be triggered by receptor binding. Identification of the domains of the para myxovirus attachment and fusion proteins that mediate membrane fusion activities is an essential part of understanding the mechanism of fusion. As a result of the H-F interaction prior to receptor binding, MV H attachment to its cellular receptor must result in conformational changes that trigger activation of the F protein. Site-directed mutagenesis analyses of two regions of MV H indicate that a HR domain in the stalk of the attachment protein is essential to the ability of H to activate F. However, either it is not the only region of H that interacts with F or it is indirectly involved in F activation because mutations in the HR do not disrupt MV H-F complex formation at the cell surface. Additionally, the functional interaction between MV H and F may be mediated, at least in part, by Loop 1 of the amino terminus of the C-rich region of the fusion protein. However, the exact role of this region of the F protein in fusion promotion remains to be determined. Importantly, the cell surface interaction between MV H and F proteins appears to be mediated by more that one region of each protein. In contrast to NDV, in no case has a definitive link between any single amino acid difference in MV H or F and an inability to form the cell surface H-F complex been established. In conclusion, the data presented in this dissertation support a model of measles membrane fusion in which the Hand F proteins form a complex prior to receptor recognition. This complex may hold F in its meta-stable pre-fusion state until binding of H to receptors at the cell surface triggers dissociation of the complex, releasing F to assume its fusogenic form. Importantly, these data also indicate that, although paramyxoviruses may all use the same general process. for promotion of membrane fusion, the mechanism may vary in multiple aspects. A more complete understanding of the means by which measles promotes membrane fusion may direct the development of specific strategies aimed at interfering with the early stages of infection.

Age stratified seroepidemiological studies play a crucial role in the design and assessment of vaccination strategies. An existing multiplex bead immunoassay for measles, mumps, rubella and varicella zoster virus antibodies together with a newly developed multiplex bead immunoassay for herpes simplex virus type 1 and type 2 antibodies were used to investigate the age-related seroepidemiology of these viruses in England during 2012.To develop the HSV-1 and HSV-2 antibody assay, attempts were made to produce full length of HSV-1 and HSV-2 glycoprotein G using a baculovirus vector expression system. While HSV-1 gG protein was produced, the proteins were extensively aggregated. Native glycoprotein G molecules undergo partial removal of HSV-1 signal sequence and HSV-1 short membrane anchor sequence during post translational modification. It is possible that such post translational modification is not performed when protein is processed in insect cell culture. Attempts to produce an HSV-2 glycoprotein G were not successful. It is possible that the high GC-content of HSV-2 glycoprotein G led to poor fidelity of copying the PCR amplification sequence. Commercially available truncated HSV-1 gG and HSV-2 gG were therefore used to develop a duplex microbead immunoassay for the simultaneous detection of specific HSV antibodies in human sera. The resultant assays performed with low sensitivity and specificity (HSV-1 of 89% and 66%, respectively and for HSV-2 of 79% and 85%, respectively) compared to the reference HerpeSelect ELISA.The MMRV multiplex bead immunoassay proved rapid, and required minimal sample volume to semi-quantify MMRV specific antibodies. The seroepidemiology of MMR results was compared with previous seroepidemiological studies performed in 1996 in England. The comparison showed an increase in the proportion of individuals who were positive for mumps and measles antibodies in the 2012 survey. The proportion of individuals positive for rubella was essentially unchanged. The increase in the proportion of individuals positive for mumps and measles antibodies in 2012 show the effectiveness of the change in MMR vaccination policy for England from 1996 onward. For VZV, the proportion of individuals who were positive for varicella antibodies between the 1996 and 2012 serological surveys were essentially unchanged. The comparison showed that most young children are susceptible to VZV. At this level of immunity, it can be expected that varicella will continue to produce epidemics of infection in the population, unless varicella vaccination is implemented as a part of routine childhood vaccination.

Les gens infectés par la rougeole subissent une suppression immunitaire. Ce mémoire porte sur l’influence de cette caractéristique de la rougeole sur un autre pathogène, ici la grippe. Il s’agit donc de modéliser la réponse immunitaire d’une interaction virus-virus, problème d’une grande pertinence durant la pandémie causée par le SRAS-CoV-2 alors que les interactions de celui-ci avec le virus de l’influenza demeurent à déterminer. Nous avons également que la grippe augmente la production d’autres pathogènes. Un modèle de chaque pathogène va être développé et analysé. On cherchera les points fixes, leurs conditions de stabilité et on observera quelques résultats numériques pour constater leurs évolutions dans le temps. Ensuite un modèle suivant l’évolution des deux pathogènes ayant infecté un individu au même moment sera conçu. Dans ce modèle nous inclurons les interactions d’un pathogène l’un sur l’autre pour déterminer théoriquement les effets chez les individus infectés à la fois par la grippe et la rougeole. On pourra par la suite comparer entre les différentes populations lorsqu’il n’y a aucune interaction et avec les différentes interactions entre les deux pathogènes. / People infected with measles experience immune suppression. This work focuses on the influence of this characteristic of measles on another pathogen, here the flu. We also have that the flu will increase the production of other pathogens in a co-infection model. Modeling the immune response to such virus-virus interaction is currently of signficant relevance, given the limited knowledge on SARS-CoV-2/influenza interactions. A model of each pathogen will be developed and analysed. We will look for the fixed points, conditions for their stability and we will observe some numerical results of their evolution over time. Then a model following the evolution of the two pathogens having simultaneously infected an individual will be designed. In this model we will include the interactions of the pathogens on each other to theoretically determine the effects in individuals infected with both influenza and measles. Then we can compare between the different populations when there is no interaction and with the different interactions between the two pathogens.

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

As the world observes a new pandemic with COVID-19, it is clear that pathogens can spread rapidly and without recognition of borders. Outbreaks will continue to occur, and so the diseases’ transmission method must be thoroughly understood in order to minimize their impact. Some infections, such as influenza, tuberculosis and measles are known to be spread through droplets in the air. In a confined space the concentration can grow as more droplets are released. This study examined a simulated confined space modelled as a hospital waiting area, where people who could have underlying conditions congregate and mix with potentially infectious individuals. It further investigated the impact of the volume of the waiting area, the number of people in the room, the placement of them as well as their weight. The simulation is an agent-based model (ABM), a computational model with the purpose of analysing a system through the actions and cumulative consequences of autonomous agents. The presented ABM features embodied agents with differing body weights that can move, breathe and cough in a ventilated room. An investigation into current epidemiological models lead to the hypothesis that one may be implemented as a corresponding ABM, where it could possibly also be improved upon. In this paper, it is shown that all parameters of the Gammaitoni and Nucci model can be taken into account in an ABM via the MASON library. In addition, proof is produced to suggest that some flaws of the epidemiological model can be mended in the ABM. It is demonstrated that the constructed model can account for proximity between susceptible people and infectors, an expressed limitation of the original model. / När världen observerar en ny pandemi, COVID-19, är det tydligt att patogener kan spridas fort och utan hänsyn till landsgränser. Utbrott kommer att fortsätta ske och därför måste sjukdomarnas överföringsmetod förstås, så att deras påverkan kan minimeras. Det är känt att vissa infektioner, såsom influensa, tuberkulos och mässling kan spridas via droppkärnor i luften. I ett begränsat utrymme kan koncentrationen växa när fler droppar tillförs. Denna studie utvärderar ett simulerat begränsat utrymme modellerat som ett väntrum på ett sjukhus, där människor som kan ha underliggande sjukdomar samlas och beblandar sig med potentiellt smittsamma individer. Inverkan av volymen av väntrummet, antalet personer i rummet, var de var placerade i rummet samt deras vikt undersöktes också. Simuleringen är en agent-baserad modell (ABM), en beräkningsmodell med syftet att analysera ett system genom handlingarna och kumulativa konsekvenserna av självstyrande agenter. Personer med olika kroppsvikt som kan röra sig, andas och hosta i ett ventilerat rum simuleras i denna ABM. Efterforskning av aktuella epidemiologiska modeller leder till hypotesen att en sådan skulle kunna implementeras som en motsvarande ABM, där den möjligtvis också kan förbättras. I denna rapport kommer det att uppvisas att alla parametrar av Gammaitonioch Nucci-modellen kan tas hänsyn till i en ABM via MASON biblioteket. Därtill produceras bevis som pekar på att vissa brister i den epidemiologiska modellen kan hämmas i denna ABM. Det demonstreras att den konstruerade modellen kan beakta distansen mellan mottagliga personer och smittsamma, vilket är en känd begränsning i originalmodellen.

A infecção é causa de morbimortalidade no paciente com insuficiência renal crônica (IRC), facilitada pela uremia, que leva a resposta imune insuficiente, inclusive após a vacinação. Foram avaliadas a situação vacinal e a presença de anticorpos para sarampo e varicela, de 83 crianças e adolescentes com IRC. A adesão dos pacientes às vacinas foi BCG 100%, poliomielite 98,8%, DPT 97,6%, sarampo monovalente 96,4%, tríplice viral 88%, hepatite B 68,7%. Ausência de anticorpos para sarampo e varicela ocorreu em 14,5% e 26,5% dos pacientes. A susceptibilidade ao sarampo em vacinados, predominou acima de seis anos (P < 0,00001) e à varicela (infecção natural) abaixo de sete anos (P < 0,001). O renal crônico pediátrico deve receber esquema vacinal amplo, com avaliação periódica de títulos de anticorpos / Infections are a cause of morbidity and mortality in chronic renal failure (CRF) patients, facilitated by uremia, which promotes a deficient immune response and hinders response to vaccination. We evaluated the immunization status and antibody titers against measles and varicella in 83 CRF children and adolescents. Adhesion to vaccination was 100% BCG, 98,8% poliomyelitis, 97,6% DPT, 96,4% measles, 88% MMR, 68,7% hepatitis B. Non-detectable antibodies against measles and varicella occurred in 14,5% and 26,5% patients. Susceptibility to measles, after vaccination, increased above 6 years (P < 0,00001) and to varicella (natural infection), below seven years of age (P < 0,001). Pediatric CRF patients should receive a robust immunization program with periodic antibody titer assessment

A infecção é causa de morbimortalidade no paciente com insuficiência renal crônica (IRC), facilitada pela uremia, que leva a resposta imune insuficiente, inclusive após a vacinação. Foram avaliadas a situação vacinal e a presença de anticorpos para sarampo e varicela, de 83 crianças e adolescentes com IRC. A adesão dos pacientes às vacinas foi BCG 100%, poliomielite 98,8%, DPT 97,6%, sarampo monovalente 96,4%, tríplice viral 88%, hepatite B 68,7%. Ausência de anticorpos para sarampo e varicela ocorreu em 14,5% e 26,5% dos pacientes. A susceptibilidade ao sarampo em vacinados, predominou acima de seis anos (P < 0,00001) e à varicela (infecção natural) abaixo de sete anos (P < 0,001). O renal crônico pediátrico deve receber esquema vacinal amplo, com avaliação periódica de títulos de anticorpos / Infections are a cause of morbidity and mortality in chronic renal failure (CRF) patients, facilitated by uremia, which promotes a deficient immune response and hinders response to vaccination. We evaluated the immunization status and antibody titers against measles and varicella in 83 CRF children and adolescents. Adhesion to vaccination was 100% BCG, 98,8% poliomyelitis, 97,6% DPT, 96,4% measles, 88% MMR, 68,7% hepatitis B. Non-detectable antibodies against measles and varicella occurred in 14,5% and 26,5% patients. Susceptibility to measles, after vaccination, increased above 6 years (P < 0,00001) and to varicella (natural infection), below seven years of age (P < 0,001). Pediatric CRF patients should receive a robust immunization program with periodic antibody titer assessment

Le syndrome fébrile, une des principales causes de consultation dans les services de pédiatrie en Afrique Subsaharienne, reste dans la majorité des cas liée à une maladie infectieuse (parasitaire, virale, bactérienne). Dans cette thèse, nous avons identifié les agents infectieux non bactériens responsables de la survenue des fièvres chez l'enfant afin de développer des outils moléculaires permettant l'amélioration de la surveillance épidémiologique. Pour ce faire, ce travail est divisé en 2 parties essentielles. Dans la première partie une étude transversale a été réalisée pour analyser l'infection plasmodiale dans une population d'adultes et d'enfants fébriles par microscopie et PCR.Dans la deuxième partie une autre enquête transversale a été conduite pour déterminer les principales causes étiologiques des fièvres non bactériennes chez l'enfant. Sur 203 enfants recrutés, 111 ont été diagnostiqués positifs à P. falciparum, par microscopie et par PCR (ISM). Concomitamment, des cas cliniques d'oreillons et de rougeole ont été observés respectivement sur les 203 enfants fébriles. Le génome complet de la souche responsable d'oreillons a été séquencé et compte 15263 nucléotides. Enfin, le virus responsable de la rougeole a été détecté par PCR et le génotypage a révélé que cette souche était celle qui était responsable de l'épidémie de Libreville.En conclusion, le syndrome fébrile chez l'enfant à Franceville est essentiellement dû aux infections à P. falciparum et Paramyxovirus. Ces résultats montrent que les infections submicroscopiques (ISM)à P. falciparum peuvent non seulement servir de réservoir mais aussi initier une symptomatologie sévère chez l'enfant. / Febrile syndrome, the main cause of consultation in pediatric wards from Sub-Saharan Africa remains in great majority associated with infectious diseases (parasites, viruses, bacteria). In this thesis, we identified the infectious agents associated with childhood fever in order to develop suitable molecular tools allowing the epidemiological surveillance. This work is divided into two main parts. Firstly, we analyzed the prevalence of Plasmodium infection in febrile patients (children and adults) by combined microscopy and PCR to determine the rate of P. falciparum submicroscopic infection (SMI).Secondly, another cross-sectional survey was conducted at pediatric ward of HASG in which the main etiological causes of febrile illness in children were investigated. Of 203 children recruited, 111 were diagnosed positive for P. falciparum by microscopy and PCR (SMI). Concomitantly, clinical cases of Mumps and Measles viruses were diagnosed respectively. The whole genome of mump virus strain isolated has been sequenced and composed of 15,263 nucléotides. Finally, Measles virus has been diagnosed by PCR and genetic analysis revealed that this strain associated with the outbreak of Libreville. In conclusion, febrile syndrome in childhood at Franceville is essentially caused by P. falciparum and Paramyxovirus infections. These results show that submicroscopic infection of P. falciparum can serve as a reservoir and also able to initiate a severe symptomatology in children.

Le transfert de gènes dans les cellules souches hématopoïétiques par des vecteurs lentiviraux s’inscrit dans les protocoles actuels de traitement par thérapie génique de plusieurs maladies monogéniques (B-thalassémie, Adrénoleucodystrophie, SCID…). De même, le transfert de gènes dans les lymphocytes T et B ouvre des perspectives tant au niveau de la thérapie génique que pour l’immunothérapie. Nous avons mis au point des vecteurs lentiviraux pseudotypés par des glycoprotéines chimérique (BaEV/TR) et mutante (BaEVRLess) du rétrovirus endogène de babouin. Nous avons montré que ces nouveaux vecteurs peuvent transduire de manière plus efficace les cellules souches hématopoïétiques stimulées et quiescentes que les vecteurs pseudotypés par la glycoprotéine du virus de la stomatite vésiculaire (VSV-G). Il en est de même pour les vecteurs développés récemment et pseudotypés par les Glycoprotéines H et F du virus de la rougeole. Nous avons aussi comparé la capacité de ces derniers vecteurs à ceux pseudotypés par les glycoprotéines BaEV/TR et BaEVRLess dans le transfert de gènes dans les lymphocytes B et T ainsi que dans l’ensemble des cellules de la lignée T. Nous sommes désormais en mesure de proposer des vecteurs adaptés au transfert de gènes à chaque étape de la différenciation des cellules CD34+ en thymocytes ainsi qu’en lymphocytes T matures. Ceci pourrait permettre de proposer de nouveaux protocoles cliniques en thérapie génique avec une co-transplantation de cellules souches génétiquement modifiées et de cellules T différenciées à partir de ces cellules. Ceci permettrait notamment de réduire les phases d’aplasie actuellement nécessaires pour la greffe de cellules souches. / Lentiviral vectors and their ability to transfer gene into hematopoietic stem cells are currently evaluated for the cure of several single-gene diseases (eg : B-thalassemia, Adrenoleucodystrophy, SCID). Likewise, gene transfer into B and T lymphocytes is of major interest in gene therapy and immunotherapy. We engineered new lentiviral vectors pseudotyped by some chimeric (BaEV/TR) and mutant (BaEVRLess) glycoproteins from the baboon endogenous retrovirus. We demonstrated that these new vectors can transduce more efficiently resting and mild stimulated hematopoietic stem cells than obtained with lentivectors pseudotyped by the glycoprotein G from the vesicular stomatitis virus (VSV-G). It is the same with the recently developed lentiviral vectors pseudotyped by the H and F glycoprotein from measles virus (H/F-LVs). We also compared the ability of the H/F-LVs with the BaEV/TR and BaEVRLess lentiviral vector pseudotype to transfer genes into B and T lymphocytes and into the whole T lineage. From now on, we are able to propose adapted vectors for gene transfer at each stage of differentiation from CD34+ cells to thymocytes and mature T cells. This could allow us to propose some new clinical protocols in gene therapy with a co-transplantation of genetically modified stem cells and their differentiated T progenitors in order to reduce the aplasia stage induced by current transplantation protocols.