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

Controlling influenza A virus (IAV) infections remains a challenge largely due to the high replication and mutation rates of the virus. IAV is a negative-sense RNA virus with two main surface proteins — hemagglutinin (HA) and neuraminidase (NA). HA recognizes and binds sialic acid on host cell receptors to initiate virus entry. NA also recognizes sialic acid on host cell receptors but functions by cleaving sialic acid interactions to release progeny virus. Because both HA and NA interact with sialic acid on the host cell surface with opposing effects, their balance is essential for optimal viral infectivity. However, the evolutionary constraints that maintain HA and NA function, while conserving a functional balance, are not fully understood. I adapted the comprehensive and systematic mutational scanning technology, termed EMPIRIC (Exceedingly Meticulous and Parallel Investigation of Randomized Individual Codons), to investigate the local fitness landscape of regions of HA under standard conditions and under drug pressure. We observed that synonymous substitutions had a higher mean absolute fitness effect in the signal than a neighboring HA region used as a control. Folding ∆G calculations revealed a hairpin loop that appeared to be differentially enriched between human and swine IAV variants in sequences of circulating strains. However, the molecular mechanism resulting in the observed host species-specific constraints remains undefined. Studying the fitness landscape of the receptor binding site of HA revealed the high sensitivity of this region to mutation. However, modulating the levels of NA activity by mutation and by using the NA inhibitor oseltamivir enabled the identification of HA mutations with adaptive potential under selection pressure by oseltamivir. These results highlight the importance of the HA-NA functional balance virus replication and in the development of resistance to oseltamivir inhibitors. These studies provide improved understanding of IAV biology, and can inform the development of improved antiviral agents with reduced likelihood for resistance.

No description available.

Traditional functional assays such as hemagglutination inhibition (HAI) and micro-neutralization (MN) assays have been routinely used for assessing the vaccine response, since influenza vaccine has been administered in people (1940). Such assays are not always predictive regarding the protection conferred by the influenza vaccine and are not able to monitor neutralization related to stem region of influenza hemagglutinin responsible for virus membrane fusion in the endosomes. In order to study Influenza vaccine response in a more biomimetic manner and overcome the deficiencies of the traditional functional assays, we developed a fluorescent membrane fusion assay (fMF). The assay uses viruses labeled with Octadecyl Rhodmaine B Chloride (R18) to monitor two major neutralization pathways: blocking the attachment of virus to the target cells and blocking of virus membrane fusion in the endosomes. The latter was tested using endosomal acidification inhibitor Bafilomycin a1 which blocked membrane fusion by 85%. Specificity of the assay was tested using two different subtypes of viruses H1N1 (A/Puerto Rico/8/1934 and A/Brisbane/59/2007), and H3N2 virus (A/Aichi/68) with their respective subtype specific stem specific monoclonal antibodies: M145, Aca-1, Aca-2 (H1N1 specific) and Aca-3 (H3N2 specific). Subtype specific mAbs blocked membrane fusion, while a mismatch in virus subtype and the mAb resulted in lack of blocking. We also studied the effect of H1N1 head specific mAb Aca-4, which not only blocked attachment of the virus, but also demonstrated blocking of membrane fusion. Results were validated by testing pre- and post- sera from 2009 seasonal Influenza vaccination and to show that at higher Ab concentration the majority of virus (85%) was blocked from attaching cells, but at lower Ab concentration, where attachment could not be prevented, blocking of membrane fusion was still in effect - up to 50%. Sera screening experiments showed that sera antibodies work beyond just blocking attachment. They also may neutralize the already attached virus by blocking fusion of the viral membrane in the endosomes. The assay has the capacity to monitor blocking of attachment and fusion in a single run. Therefore, it is more representative regarding the natural process of infection and the corresponding neutralization pathways. The assay is unique in terms of assessing stem specific antibodies; stem specific response and its measurement are relevant for the advancement of a universal influenza vaccine.

No description available.

Die Fusion von Membranen erfordert die Verschmelzung von zwei Phospholipiddoppel-schichten, wobei dies über dieselben Zwischenschritte abzulaufen scheint. Eine lokale Störung (‚Stalk’) stellt eine erste Verbindung der äußeren Membranhälften dar, die anschließend lateral expandiert und ein Hemifusionsdiaphragma (HD) bildet. Das Öffnen einer Fusionspore im HD führt zur vollständigen Fusion. Mittels konfokaler Mikroskopie wurde die Fusion von Giant unilamellar vesicles (GUVs) mit negativ geladenen Lipiden und transmembranen (TM) Peptiden in Anwesenheit von zweiwertigen Kationen beobachtet, wobei die Peptide bei der HD Entstehung völlig verdrängt wurden. Eine detaillierte Analyse zeigte, dass es sich bei diesem Mikrometer-großen Bereich um ein HD handelt, dessen Größe von der Lipidzusammensetzung und Peptidkonzentration in den GUVs abhängt. Laterale Lipiddomänen gelten als entscheidend für Signal- und Sortierungsprozesse in der Zelle. Liquid ordered (Lo) Domänen in Modellsystemen wie GUVs ähneln den mit Sphingo-lipiden und Cholesterol angereicherten biologischen Raft-Domänen, allerdings scheinen Membraneigenschaften wie die Lipidpackung sich von biologischen Membranen zu unterscheiden. In diesem Zusammenhang wird die Sortierung des TM-verankerten Hemag-glutinin (HA) des Influenzavirus und von lipidverankerten Ras-Proteinen in GUVs wie auch in abgelösten Plasmamembran-Ausstülpungen (GPMVs) untersucht. HA Protein und TM-Pepitde von HA wurden ausschließlich (GUVs) bzw. vorwiegend (GPMVs) in der liquid disordered (Ld) Domäne gefunden. K-Ras wurde inmitten der Ld detektiert, während N-Ras zur Lo/Ld Grenzlinie diffundierte. Diese Ergebnisse werden im Zusammenhang mit den Unterschieden der Lipidpackung innerhalb der verschiedenen membranverankerten Systeme diskutiert. Es ist wahrscheinlich, dass die Bildung, Größe und Stabilität sowie die physikalischen Eigenschaften der Lipiddomänen in biologischen Membranen stark von Protein-Lipid-Wechsel-wirkungen beeinflusst werden. / Membrane fusion is ubiquitous in life and requires remodelling of two phospholipid bilayers. Fusion likely proceeds through similar sequential intermediates. A stalk between the contacting leaflets forms and radially expands into a hemifusion diaphragm (HD) wherein finally a fusion pore opens up. Direct experimental verification of this key structure is difficult due to its transient nature. Confocal microscopy was used to visualize the fusion of giant unilamellar vesicles (GUVs) comprising negatively charged phosphatidylserine and fluorescent transmembrane (TM) entities in the presence of divalent cations. A complete displacement of TM peptides preceded full fusion. This is consistent with HD formation. Detailed analysis provided proof that the micrometer sized structures are in fact HDs. HD size is dependent on lipid composition and peptide concentration. Lateral lipid domain formation is believed to be essential for sorting and signalling processes in the cell. Liquid ordered (Lo) domains in model systems like GUVs resemble biological rafts enriched in sphingolipids and cholesterol, but their physical properties seem distinct from biological membranes as judged by e.g. lipid order and packing. In this context the sorting of TM anchored influenza virus hemagglutinin (HA) and different lipid anchored Ras proteins is studied in GUVs and giant plasma membrane derived vesicles (GPMVs). Authentic HA or the TM domain peptides were sorted exclusively (GUVs) or predominantly (GPMVs) to the liquid disordered (Ld) domains. Whereas K-Ras was found in the bulk Ld domains, N-Ras diffuses to the Lo/Ld interface. These results are discussed with respect to differences in lipid packing in the different membrane systems and regarding the membrane anchors and their hydrophobic matching. The results suggest that the formation, size and stability as well as the physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.

Mycobacterium avium ssp. paratuberculosis (Map), agent étiologique de la paratuberculose, a évolué en deuxtypes dénommés, S pour« Sheep » et C pour « Cattle ». L’intestin grêle est le site primaire de l’infection à Map mais les mécanismes moléculaires impliqués dans l’implantation du bacille restent largement méconnus. L’objectif de mon projet de thèse visait à identifier et caractériser les adhésines exprimées par Map par des approches génétiques et biochimiques. J’ai ainsi purifié la HBHA et la LBP par chromatographie d’affinité puis les ai identifiés en spectrométrie de masse. L’originalité de ce travail repose sur le polymorphisme de ces adhésines observé entre les souches de type C et S. Cette variabilité a été mise en évidence sur le domaine d’interaction avec les sucres sulfatés de la cellule hôte influençant l’affinité des adhésines pour l’héparine. Ce travail de thèse a permis de caractériser pour la première fois ces deux adhésines produites par Map. Le polymorphisme de la HBHA et de la LBP, discriminant les types C et S, ouvre de nombreuses perspectives sur l’évolution de l’espèce M. avium et le rôle de ces adhésines sur le tropisme intestinal, la préférence d’hôte de Map ou encore leur potentiel diagnostic. / Mycobacterium avium subsp. paratuberculosis (Map), the etiological agent of paratuberculosis, has evolved into two types called, S for "Sheep" and C for "Cattle." The small intestine is the primary site of Map infection but the molecular mechanisms involved in the establishment of bacilli are still unknown. The aim of my thesis was to identify and characterize the adhesins expressed by Map by genetic and biochemical approaches. I purified HBHA and LBP by affinity chromatography then identified them by mass spectrometry. The originality of this work is based on the polymorphism of these adhesins observed between strains of type C and S. This variability has been demonstrated in the binding domain involved in interaction with sulfated sugars of host cell influences adhesins affinity for heparin. This thesis has characterized for the first time these two adhesins produced by Map. Specific polymorphism highlighted related to the evolution of the species avium, opens large number questions on their role on the pathogenesis of Map including the cellular tropism, host preference or interest of these antigens to improve diagnostic.

No description available.

Es wird angenommen, dass das Influenzavirus-Glykoprotein Hämagglutinin (HA) für seine Funktion sowohl bei der Virusfreisetzung als auch bei der Fusion von viraler und zellulärer Membran mit Cholesterin- und Sphingolipidreichen Domänen, sogenannten Membran-Rafts, assoziiert sein muss. Aus diesem Grund sollte in dieser Arbeit die Membran-Raft-Affinität von HA in lebenden Zellen mittels FLIM-FRET gemessen werden. Dabei wurde mit Hilfe der Fluroreszenz-Lebenszeit-Messung (FLIM) der Förster-Resonanz-Energie-Transfer (FRET) von fluoreszenzmarkiertem HA auf einen etablierten Raft-Marker bestimmt. Diese Messungen zeigten, dass beide Proteine in gemeinsamen Klustern in der Plasmamembran vorkommen. Durch Cholesterinentzug und durch den Einsatz von Cytochalasin D, welches die Mikrofilamente zerstört, konnte diese Klusterbildung reduziert werden. Demnach tragen sowohl die Membran-Rafts als auch das Aktinnetzwerk zu dieser Klusterbildung bei. Mittels FLIM-FRET konnte zusätzlich bestätigt werden, dass die Signale für die Detergenslöslichkeit von HA in Triton-Extraktionsexperimenten, die Palmitylierung und die stark hydrophoben Aminosäuren zu Beginn der Transmembrandomäne (TMD), auch im lebenden System eine wichtige Rolle spielen. Zusätzlich konnten biochemische Experimente zeigen, dass die hydrophoben Aminosäuren zu Beginn der HA-TMD den intrazellulären Transport, nach der Trimerbildung, entscheidend verzögern. Diese Verzögerung ist vermutlich auf einer erschwerten Integration dieser Proteine in die Membran-Rafts begründet. Die virale Fusion mit der Wirtszellmembran wird durch eine pH5-Behandlung vermittelte Konformationsänderung von HA ausgelöst. FLIM-FRET-Messungen zeigten für die pH5-Konformation von HA eine verglichen mit der pH7-Konformation verringerte Klusterbildung mit dem Raft-Marker. Somit ist offensichtlich, dass die Membranfusion-vermittelnde HA-Konformation eine verringerte Raft-Affinität besitzt. Diese verringerte Raft-Affinität könnte eine wichtige Rolle bei der Störung der Lipide an der Fusionsstelle spielen und somit die Bildung und/oder Vergrößerung der Fusionspore erleichtern. / It has been supposed that the hemagglutinin (HA) of influenza virus is recruited to cholesterol- and sphingolipid-enriched domains, also named membrane-rafts, to accomplish its function in virus budding and membrane fusion. This study aimed at verifying the affinity of HA for membrane-rafts in living cells using fluorescence-lifetime imaging microscopy to measure Förster’s resonance energy transfer (FLIM-FRET). FLIM-FRET revealed strong clustering between a fluorescence-tagged HA-protein and a well-established raft-marker in CHO cells. Clustering was significantly reduced when rafts were disintegrated by cholesterol depletion and when microfilaments were disrupted with cytochalasin D. Thus, membrane-rafts as well as the actin meshwork contribute synergistically to clustering. Clustering was also reduced by the removal of the known signals for the association of HA with detergent-resistant-membranes, the palmitoylation and the first amino acids in the transmembrane region (TMR). Since these mutations are obviously important for the raft-association of HA their function during the transport through the ER and the Golgi-complex was studied. These investigations showed that the exchange of the first three amino acids of the HA-TMR led to a decelerated transport after trimer-formation of the protein, probably due to retarded integration of these proteins into membrane-raft domains. Mediating viral fusion with the host cell membrane requires an irreversible conformational change of HA. FLIM-FRET studies of this low pH conformation unveiled that the clustering with the raft-marker is decisively reduced compared to the pre-fusion conformation of the protein. It might be assumed that the fusion-mediating conformation of HA reduces the proteins affinity for membrane-rafts. Therefore it is likely that this reduced affinity for rafts after the conformational change is relevant to cause perturbation of lipids at the fusion site and thereby facilitating the formation and/or enlargement of the fusion pore.

Zusammenfassung Zur Freisetzung ihres Genoms in das Innere der Wirtszelle müssen Hüllviren ihre Membran mit der Membran der Wirtszelle verschmelzen. Diese Fusion wird durch eine Konformations- umwandlung der Ektodomäne viraler Glykoproteine ausgelöst. Die Kenntnis der drei- dimensionalen Struktur der vollständigen Ektodomäne in der fusionsaktiven Konformation ist Voraussetzung für das Verständnis des Fusionsmechanismus. Die Fusion von Influenza mit entsprechenden Targetmembranen wird durch eine durch sauren pH ausgelöste Konformationsänderung des als Trimer vorliegenden Glykoproteins Hämagglutinin (HA) vermittelt. Bis jetzt war die dreidimensionale (Röntgenkristall-) Struktur einer enzymatisch abgespaltenen HA-Ektodomäne nur eines Influenzastammes bei neutralem pH-Wert bzw. von einigen Fragmenten der HA2-Untereinheit bei saurem pH-Wert bekannt. In der vorliegenden Arbeit wurden ein geeigneter Influenzastamm sowie geeignete Untersuchungsbedingungen ermittelt, um die 3D-Struktur des kompletten, nicht enzymatisch vorbehandelten HA sowohl in seiner nativen (bei neutralem pH-Wert vorliegenden) als auch in seiner fusionskompetenten (keinesfalls aber bereits inaktivierten) Struktur mittels Kryo- Elektronenmikroskopie und Bildverarbeitung aufzuklären. Es wurde erstmals die 3D-Struktur des kompletten HA eines anderen Influenzastammes (A/Japan) bei neutralem pH-Wert aufgeklärt und mit der bekannten 3D-Struktur von Influenza X-31 verglichen. Außerdem konnte eine fusionskompetente Form rekonstruiert werden, die im Vergleich zur nativen Konformation deutliche Veränderungen in der 3D-Struktur zeigt, ohne daß sich jedoch die Assoziation der Monomere aufhob. Die Befunde werden unter anderem in Hinblick auf die Bedeutung der HA1-Untereinheit für die Fusion diskutiert. Die vorgestellte Methode scheint geeignet, auch andere Membranproteine bzw. Membranfusion-vermittelnde Proteine in verschiedenen konformeren Zuständen aufzuklären (insbesondere jene, die der Röntgenkristallstrukturanalyse nicht zugänglich sind) und so einen diesen Fusionsprozessen eventuell zugrundeliegenden konservierten Funktionsmechanismus aufzuhellen. / Abstract Envelope viruses enter a host cell via fusion between the viral and endosomal membrane, thereby releasing the nucleocapsid into the cytoplasm. The fusion has been accompanied with an conformational change in the ectodomain of viral glycoproteins. To understand the mechanism leading to fusion the three-dimensional (3D) structure of the complete viral glycoprotein in its fusion competent conformation has to be determined. The Fusion of influenza virus which is triggered at low pH has been associated with an irreversible conformational change in the trimeric glycoprotein hemagglutinin (HA). The three-dimensional (crystal) structure is already known of the enzymatic-cleaved ectodomain of one influenza strain (X-31) at neutral pH or of some fragments of the HA2-subunit at low pH, respectively. In this work a suitable influenza strain as well as suitable experimental conditions for investigations of the 3D-structure of the complete and not enzymatically treated HA at neutral and acidic pH conditions have been determined. An cryo-microscopy/angular reconstitution approach has been employed for the 3D- reconstruction of the intact HA of an different influenza strain (A/Japan). This structure is in excellent agreement with the known X-ray crystallographic structure of the bromelain- cleaved ectodomain of HA from influenza X-31. Moreover, for the very first time the 3D structure of the intact HA of Influenza A/Japan in its fusion competent state (at acidic pH) has been calculated. The differences between the two structures are large compared to the marginal differences between the neutral pH structures by EM and by X-ray crystallography, respectively, although the monomers remain tightly connected. These findings will be discussed especially with regard to the role of the HA1 subunit in the fusion process. The procedure is in general applicable to pursue the 3D-structure of (fusion mediating) proteins in various conformational states (especially of those proteins which are not directly accessible by X-ray crystallography). This approach should offer to elucidate the (eventually conserved) mechanism of membrane fusion.