The Australian paralysis tick, Ixodes holocyclus causes a potentially fatal paralysis in domestic animals, livestock and humans with companion animals (mainly dogs) most commonly affected. Current treatment regimes include administration of a commercial tick anti-serum (TAS), prepared as hyperimmune serum in dogs, to neutralise the effects of the toxin. However, each new batch must be standardised using an expensive and highly subjective bioassay performed in neonatal mice. There is currently an urgent need for a more cost effective and rapid in vitro assay that can be more objectively and accurately quantified. Further understanding of the composition of the toxin molecule is also required to develop toxin-specific reagents necessary for these assays. One of the main objectives of this study was to develop a suitable immunoassay to replace the existing mouse bioassay for assessing batches of tick anti-sera for use in tick paralysis therapy in dogs. Initially an enzyme-linked immunosorbent assay (ELISA) was established to detect and quantify antibody specific for I. holocyclus toxin in dog sera. Using a partially purified antigen extracted from I. holocyclus salivary glands, good discrimination was achieved between reactive (hyperimmune) and non-reactive (naïve) sera. The hyperimmune dog sera reacted very strongly with the antigen compared to negligible reactions of serum from dogs not exposed to I. holocyclus. The reactions of hyperimmune sera were also significantly weaker to a non-toxin antigen control extracted from the salivary glands of the non-toxic tick Rhipicephalus microplus, indicating the assay was detecting toxin-specific responses. Furthermore, each of the hyperimmune sera that reacted strongly and specifically with the I. holocyclus antigen in the ELISA also strongly neutralised toxin in the mouse bioassay. Together these findings support the suitability of this ELISA for assessing the potency of batches of commercial dog hyperimmune sera for use as therapy for tick paralysis in dogs. Sera from dogs that were experimentally infested with ticks and sera from patient dogs, presenting at veterinary clinics with signs of tick paralysis, were also screened for antibodies to I. holocyclus antigen using the ELISA. Twenty-eight out of 29 sera from animals with single or multiple exposures to ticks failed to recognise the I. holocyclus antigen indicating the ELISA is not suitable as a diagnostic test to detect toxin-specific antibodies in animals with limited exposure to I. holocyclus infestation. A panel of toxin-specific monoclonal antibodies (mAbs) was produced as research tools to analyse and purify tick toxin components. Rats were successfully immunised against tick toxin using a combination of inoculation of partially purified salivary gland antigen and exposure to tick infestation. The latter approach preserved the native confirmation of the toxin using a natural route of immunisation and rats were chosen due to their high tolerance of multiple tick infestations over several days. While fusion of rat spleen cells with mouse myeloma cells has been reported several times in the literature, the resulting hybridomas are unstable with fastidious culture requirements. Optimisation of the culture conditions revealed that most rat-mouse hybridoma lines grew best in serum-free medium supplemented with 5% foetal bovine serum. Of 600 hybridomas produced, only 12 were shown to be specific for the Ixodes antigen, as determined by ELISA. A selection of these hybridomas representing various patterns of affinity and/or antigen specificity were further analysed for toxin-neutralising ability in a mouse bioassay. Notably, the most potent toxin-neutralising mAb in mice, showed a specific but relatively moderate reaction to Ixodes antigen in the ELISA. The most potent toxin-neutralising mAbs inactivated toxin as strongly as the commercial TAS used for immunotherapy in dogs with tick paralysis. This suggests that mAbs may present an alternative source of immunotherapy, providing a potentially endless supply of a highly consistent reagent and negating the need to use live animals for both the production of tick antiserum and the continual testing of reagent batches. The toxin-neutralising mAbs were also used to analyse I. holocyclus toxin in polyacrylamide gel electrophoresis (PAGE) and Western blot to identify specific toxin proteins. The most potent neutralising mAbs consistently recognised high MW proteins (100-200 kDa) in a smeared pattern. Although this was contrary to previous reports of low molecular weight components (3-5 kDa) in holocyclotoxin, this study was the first to use mAbs prepared to native toxin. The large molecular weight structures likely represent presucursors to, or complexes of the smaller peptides, previously identified. When the Toxin-neutralising mAbs were assessed as ligands to affinity purify toxin components from crude Ixodes SG extracts, toxin components of 110 and 32 kDa were consistently identified. These purified proteins represent good candidates for N-terminal sequencing to further identify the toxin components in I.holocyclus salivary glands.

Zugl.: Marburg, Universiẗat, Diss., 2007.

Les étapes précoces de l’infection virale sont des évènements critiques dans le déroulement du cycle viral. Notamment, l’entrée virale est la première étape d’interaction entre un virus et une cellule permettant d’initier, de maintenir et de propager l’infection. De ce fait, cette étape constitue une cible majeure de la réponse immunitaire adaptative de l’hôte. C’est ainsi que cette étude bipartite s’est intéressée aux étapes précoces de l’infection du virus de l’hépatite C (HCV). Dans un premier temps, l’obtention de clones de cellules Huh-7.5 n’exprimant plus le récepteur néonatal des immunoglobulines, le FcRn, a permis d’analyser l’implication de ce récepteur dans la neutralisation du HCV par des anticorps neutralisants. Les résultats obtenus nous informent que le récepteur FcRn n’intervient vraisemblablement pas dans la modulation de la neutralisation du HCV. Dans un second temps, nous avons réalisé une étude préliminaire afin d’approfondir les mécanismes régissant l’endocytose du HCV : à savoir, quelles sont les protéines adaptatrices responsables du déclenchement de l’endocytose dépendante de la clathrine et s’il existe une potentielle voie d’entrée alternative pour ce virus. A ces fins, nous avons opté pour une stratégie basée sur la transfection de siRNA, couplée à l’utilisation des pseudoparticules HCVpp qui constituent une approche encore pertinente appliquée à l’étude de l’entrée du HCV. / The early steps during a viral infection are critical events in the course of the viral cycle. Particularly, the viral entry is the first step allowing the interaction between a virus and a cell to initiate, maintain and propagate an infection. Therefore, this very step is a major target for the host adaptive immunity. This bipartite study is focused on the early steps of the infection by the hepatitis C virus (HCV). First of all, generation of Huh-7.5 clones whose expression of the neonatal Fc receptor, FcRn, has been deleted gave us the opportunity to analyze the involvement of this receptor during the neutralization of HCV by neutralizing antibodies. Regarding the results, it appears unlikely that the FcRn modulates the neutralization of HCV. Then we conducted a preliminary study to further explore the mechanisms underlying the endocytosis of HCV: that is, which are the adaptor proteins that trigger the clathrine-mediated endocytosis and if there is another putative entry pathway for the virus. To these ends we opted for a RNAi based strategy coupled to the use of HCV-derived pseudo-particles (HCVpp) that are still useful and relevant tools dedicated to HCV-entry studies.

Notre laboratoire a développé une stratégie d'isolement de fragments d'anticorps recombinants à partir de primates non humains (Macaca fascicularis) immunisés, en utilisant la technologie des phages. Dans le cadre de cette thèse, une comparaison des séquences d'anticorps de macaques (Macaca Mulatta) et d'anticorps humains a toutefois montré que les anticorps des deux espèces présentent des différences qui rendent souhaitable une étape d'humanisation des anticorps de macaques. Cette stratégie a été utilisée dans le cadre du projet Européen AntiBotABE (www.antibotabe.com) et l'étape de criblage a été adaptée pour isoler des scFv neutralisant de façon croisée les toxines botuliques BoNT/B des sous-types B1 et B2, en utilisant séquentiellement l'holotoxine BoNT/B1 et un fragment recombinant représentant la région C-terminale de la chaîne lourde de BoNT/B2. Le meilleur scFv ciblant les régions C-terminales des chaînes lourdes de BoNT/B1 et BoNT/B2, B2-7, a montré une bonne capacité de neutralisation de BoNT/B1 et BoNT/B2 dans le test ex vivo de paralysie hémidiaphragmatique. Les régions charpentes du scFv B2-7 ont un pourcentage d'identité élevé (80 %) avec leurs homologues humains. Des scFv neutralisant BoNT/A1 en ciblant sa chaîne légère ont aussi été isolés, dont le scFv le plus efficace, 2H8, induit une diminution de 50% de l'activité endopeptidasique à une concentration correspondant à un rapport molaire 2H8/BoNT/A1 de 64000. Les régions charpentes de 2H8 ont également un pourcentage d'identité élevée (88%) avec leurs homologues humains. La versatilité de cette stratégie en fait un outil permettant l'isolement de nombreux autres fragments d'anticorps à visée thérapeutique. / Our laboratory has developed a strategy to isolate recombinant antibody fragments technology from immunized non human primates (Macaca fascicularis) by phage display. In the course of the present thesis, a comparison between macaque (Macaca mulatta) and human antibody sequences has demonstrated that antibodies of the two species are different. This difference makes the humanization of macaque antibodies desirable. The strategy was used in the framework of the European AntiBotABE project, and the screening was adapted to isolate antibody fragments cross neutralizing the B1 and B2 subtypes of botulinum B neurotoxin, by using sequentially the holotoxin BoNT/B1 and a recombinant fragment representing the C-terminal region of the heavy chain of BoNTB2. The best scFv targeting the C-terminal region of BoNT/B1 and BoNTB2 heavy chains, B2-7, demonstrated a high capacity to neutralize BoNT/B1 and BoNT/B2 in the ex vivo hemidiaphragmatic assay. A high identity (80%) between the framework regions of B2-7 and their human homologs was observed. ScFvs neutralizing BoNT/A1 by targeting its light chain were also isolated and among them, the scFv 2H8 induced a decrease of 50% in the endopeptidase activity at a concentration corresponding to a molar ratio of 2H8/BoNT/A1 of 64000. A high identity (88%) between the framework regions of 2H8 and their human homologs was also observed. Our strategy can be used to isolate other therapeutic antibody fragments.

HIV est principalement transmis par voie sexuelle. Bien que les stratégies de prévention basées sur les antirétroviraux (TasP et PrEP) soient efficaces, d'autres approches (en particulier vaccinales) restent d' actualité.Dans la première partie de ce travail, nous avons caractérisé le réservoir séminal de HIV chez des patients chroniquement infectés et sous trithérapie antirétrovirale (TARV). Dans le sperme, HIV est présent sous forme libre et/ou associée aux NSMC. Ces deux formes peuvent être transmises. Le TARV réduit la quantité de virus libre dans le sperme, sans éliminer le virus associé aux NSMC. A heure du TasP, comme meilleur outil de prévention de la transmission sexuelle, mieux caractériser l'état de ce virus résiduel (latent, réplicatif, défectif ?) est nécessaire. Dans notre travail, le virus associé aux NSMC n'a pas été réactivé, laissant supposer que le virus résiduel dans les NSMC de patients sous TARV au long cours, peut être défectif. Ces données restent à confirmer dans de plus grandes cohortes.Dans la deuxième partie du travail, nous avons caractérisé sur le plan isotypique et fonctionnel, les anticorps dirigés contre HIV du sperme. L'objectif est de mettre en évidence des anticorps « large spectre » neutralisant HIV, et/ou inhibant la transcytose, et/ou capables d'ADCC et ciblant les souches virales potentiellement transmissibles par voie sexuelle. Nous avons identifié dans le plasma séminal de certains patients des anticorps neutralisant des souches X4 tropiques, alors que les souches majoritairement transmises par voie sexuelle sont R5 tropiques. Nous avons observé la capacité d' anticorps dirigés contre HIV du plasma séminal à traverser un épithélium monocouche. Le rôle protecteur ou facilitateur est à préciser. Des anticorps protecteurs pourraient être utilisés dans des formulations de type microbicides ou en immunothérapie et contribuer également à un développement de vaccin après identification des épitopes cibles. / HIV new infections are mostly related to sexual transmission. Semen contains HIV free particles and cell-associated HIV. Both forms are sexually transmitted. Although PrEP and TasP are efficient to prevent HIV sexual transmission, other strategies like a Vaccine remain needed.In a first part, we studies HIV reservoir in HIV-infected patients receiving combined antiretroviral treatment (cART) in semen. cART reduced HIV viral load in semen, but had a no activity against cell-associated HIV. We tried to reactivate HIV cell reservoir in semen to better characterize its role. We did not observe any reactivation; HIV reservoir in semen might be mostly latent, or defective. This observation should be confirmed in larger cohort of patients.In a second part, we aimed to study the prevalence of anti-HIV antibodies in semen and their role in sexual transmission. Semen of HIV-infected men contains anti-HIV immunoglobulins (Igs), mostly IgG. We observed that unspecific Igs (IgG, IgAl, IgA2) and anti-HIV IgG and IgA were able to transmigrate across an epithelium monolayer mimicking endocervix. This transmigration property may facilitate HIV transcytosis. We also observed a neutralizing activity by Igs purified from semen of 2 chronically infected patients against a X4-tropic viral strain. Seminal anti-HIV may exhibit a dual role (facilitating or limiting) HIV sexual transmission. Protective antibodies purified from semen might be used in preventive strategies like microbicides or serotherapy, and may help to develop vaccine after identification of their epitopes.

La difficulté à induire des anticorps capables de neutraliser (anticorps neutralisants, AcN) la très grande diversité des isolats circulants du VIH-1 reste à l’heure actuelle un obstacle majeur au développement d'un vaccin préventif contre le VIH-1. L’objectif du projet a été de déterminer quels étaient les épitopes neutralisants les plus conservés au sein des 4 groupes M, N, O et P de VIH-1 puis de concevoir un immunogène qui serait capable d’induire la production d’AcN anti-VIH-1. Nous avons montré que l’épitope N160-glycane dépendant de la région V1/V2 de l’enveloppe virale est le plus conservé au sein des 4 groupes du VIH-1 (Morgand et al., JAIDS 2016). Nous avons ensuite montré la faisabilité d’obtenir, en système d’expression transitoire, des particules chimères constituées de la protéine d’enveloppe HBs du virus de l’hépatite B exprimant à leur surface les glycoprotéines d’enveloppe de différents groupes et sous-type du VIH-1. Malgré la présence de certains épitopes neutralisants (supersite N332-V3, site de liaison au CD4, région MPER- membrane proximal external region-), les épitopes d’intérêt de la région V1/V2 ne sont pas exposés sur ces particules chimères. / The difficulty to induce antibodies able to neutralize (neutralizing antibodies, NAb) the large diversity of HIV- 1 isolates remains a major hurdle toward the development of an anti-HIV-1 vaccine. The aim of our study was first, to identify which epitopes are the most conserved within the 4 HIV-1 groups (M, N, O, P) and then, to design an immunogen that would be able to induce NAb against HIV-1. We showed that the V1/V2 N160- glycan epitope is the most conserved within the 4 HIV-1 groups (Morgand et al., JAIDS 2016). Subsequently, we showed the feasibility to generate chimeric particles based on the HBs envelope protein exposing the envelope glycoproteins of different groups and subtypes of HIV-1 at their surface. Although we demonstrated the presence of several neutralizing epitopes on these chimeric particles (N332-V3 supersite, CD4 binding site, membrane proximal external region), none of them exposed the V1/V2 epitopes of interest.

Yes / This paper presented a planar printed multiple-input-multiple-output (MIMO) antenna with a dimension of 100 x 45 mm2. It composed of two crescent shaped radiators placed symmetrically with respect to the ground plane. Neutralization line applied to suppress mutual coupling. The proposed antenna examined both theoretically and experimentally, which achieves an impedance bandwidth of 18.67% (over 2.04-2.46 GHz) with a reflection coefficient < -10 dB and mutual coupling minimization of < -20 dB. An evaluation of MIMO antennas is presented, with analysis of correlation coefficient, total active reflection coefficient (TARC), capacity loss and channel capacity. These characteristics indicate that the proposed antenna suitable for some wireless applications.

This thesis deals with experimental studies of electron transfer reactions between oppositely charged ions (mutual neutralisation). These were performed at the unique double electrostatic ion storage ring DESIREE at Stockholm University, which was put into full operation in 2017. In the first two published articles of this thesis, two atmospheric collision systems are treated, namely O+/O−  and N+/O−. The aim was to reproduce previous published results from a single-pass (non-stored) merged ion beams setup in UCLouvain (Belgium) and thus provide a measure of DESIREE’s capacity and resolution. In addition, the effects of metastable ions were investigated with the support of theoretical calculations. The third published paper of this thesis deals with collisions between I+ and I− (iodine ions), a process relevant to electric thrusters for new spacecraft. The results are compared with theoretical calculations in order to provide an understanding of how the reaction takes place. Preliminary results on electron transfer reactions between diatomic molecules and atoms are presented.

No / This paper presented a planar printed multiple-input-multiple-output (MIMO) antenna with a dimension of 100 x 45 mm 2 . It composed of two crescent shaped radiators placed symmetrically with respect to the ground plane. Neutralization line applied to suppress mutual coupling. The proposed antenna examined both theoretically and experimentally, which achieves an impedance bandwidth of 18.67% (over 2.04-2.46 GHz) with a reflection coefficient < -10 dB and mutual coupling minimization of < -20 dB. An evaluation of MIMO antennas is presented, with analysis of correlation coefficient, total active reflection coefficient (TARC) and capacity loss. These characteristics indicate that the proposed antenna suitable for some wireless applications. Mutual Coupling Reduction of Two Elements Antenna for Wireless Applications. Available from: https://www.researchgate.net/publication/261064207_Mutual_Coupling_Reduction_of_Two_Elements_Antenna_for_Wireless_Applications [accessed Aug 1, 2017].

Yes / The paper explores the discrepancy between attitude and behavioural intention in ethical consumption, focusing on the role of techniques of neutralisation. Drawing on findings of 251 respondents in the UK, results suggest despite positive attitude towards ethical consumption, consumers are also susceptible to the techniques of neutralisation. Hierarchical and moderated regression analyses reveal that inclusion of the neutralisation construct moderates the influences of attitudes on behavioural intention, and advances the model’s predictive capacity. In spite of suggested positive attitude towards ethical consumption, real existing behaviour is frequently filtered through the techniques of neutralisation. The sample is restricted to in size and location, however the study clearly establishes techniques of neutralisation as a construct in the decision-making process, further warranting examination of each of the techniques. Summary statement of contribution: The study confirms validity of the addition of the neutralisation construct into the modified TPB model noted by Chatzidakis et al. (2007). It suggests improvement in predicting behavioural intention and shows the moderating effects the techniques of neutralisation have on constructs in the modified TPB model. The neutralisation construct is itself found to have a significant impact on moderating purchasing intention in ethical consumption.