Antibody microarrays constitute a next-generation sensing platform that has the potential to revolutionize the way that molecular detection is conducted in many scientific fields. Unfortunately, current technologies have not found mainstream use because of reliability problems that undermine trust in their results. Although several factors are involved, it is believed that undesirable protein interactions with the array surface are a fundamental source of problems where little detail about the molecular-level biophysics are known. A better understanding of antibody stability and antibody-antigen binding on the array surface is needed to improve microarray technology. Despite the availability of many laboratory methods for studying protein stability and binding, these methods either do not work when the protein is attached to a surface or they do not provide the atomistic structural information that is needed to better understand protein behavior on the surface. As a result, molecular simulation has emerged as the primary method for studying proteins on surfaces because it can provide metrics and views of atomistic structures and molecular motion. Using an advanced, coarse-grain, protein-surface model this study investigated how antibodies react to and function on different types of surfaces. Three topics were addressed: (1) the stability of individual antibodies on surfaces, (2) antibody binding to small antigens while on a surface, and (3) antibody binding to large antigens while on a surface. The results indicate that immobilizing antibodies or antibody fragments in an upright orientation on a hydrophilic surface can provide the molecules with thermal stability similar to their native aqueous stability, enhance antigen binding strength, and minimize the entropic cost of binding. Furthermore, the results indicate that it is more difficult for large antigens to approach the surface than small antigens, that multiple binding sites can aid antigen binding, and that antigen flexiblity simultaneously helps and hinders the binding process as it approaches the surface. The results provide hope that next-generation microarrays and other devices decorated with proteins can be improved through rational design.

Influenza B virus hemagglutinin (HA) is a major surface glycoprotein with frequent amino-acid substitutions. However, the roles of antibody selection in the amino-acid substitutions of HA were still poorly understood. An analysis was conducted on a total of 271 HA 1 sequences of influenza B virus strains isolated during 1940∼2007 finding positively selected sites all located in the four major epitopes (120-loop, 150-loop, 160-loop and 190-helix) supporting a predominant role of antibody selection in HA evolution. Of particular significance is the involvement of the 120-loop in positive selection. Influenza B virus HA continues to evolve into new sublineages, within which the four major epitopes were targeted selectively in positive selection. Thus, any newly emerging strains need to be placed in the context of their evolutionary history in order to understand and predict their epidemic potential. As key epigenetic regulators, polycomb group (PcG) proteins are responsible for the control of cell proliferation and differentiation as well as stem cell pluripotency and self-renewal. To facilitate experimental identification of PcG target genes, which are poorly understood, we propose a novel computational method, EpiPredictor , which models transcription factor interaction using a non-linear kernel. The resulting targets suggests that multiple transcription factor networking at the cis -regulatory elements is critical for PcG recruitment, while high GC content and high conservation level are also important features of PcG target genes. To try to translate the EpiPredictor into human data, we performed a computational study utilizing 22 human genome-wide CHIP data to identify DNA motifs and genome features that would potentially specify PRC2 using five motif discovery algorithms, Jaspar known transcription binding motifs, and other whole genome data. We have found multiple motifs within the various subgroups of experimental categories that have much higher enrichment against CHIP identified gene promoter than among random gene promoters. Specifically, we have identified Low CpG content CpG Islands (LeG's) as being critical in the separation of Cancer cell line identified targets from Embryonic Stem cell line identified targets. Additionally, there are differences between human and mouse ES cell predictions using the same motifs and features suggesting relevant evolutionary divergence.

Although wild birds are natural hosts of avian influenza viruses (AIVs), these viruses can be highly contagious to poultry and a zoonotic threat to humans. The propensity of AIV for genetic variation through genetic shift and drift allows virus to evade vaccine mediated humoral immunity. An alternative approach to current vaccine development is induction of CD8+ T cells which responds to more conserved epitopes than humoral immunity and targets a broader spectrum of viruses. Since the memory CD8+ T lymphocyte responses in chickens to individual AIV proteins have not been defined, the modulation of responses of the memory CD8+ T lymphocytes to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins over a time course were evaluated. CD8+ T lymphocyte responses induced by intramuscular inoculation of chickens with AIV HA and NP expressing cDNA plasmids or a non-replicating human adenovirus vector were identified through ex vivo stimulation with virus infected, major histocompatibility complex (MHC) matched antigen presenting cells (APCs). The IFN? production by activated lymphocytes was evaluated by macrophage production of nitric oxide and ELISA. MHC-I restricted memory T lymphocyte responses were determined at 10 days and 3, 5, 7 and 9 weeks post-inoculation (p.i). The use of non-professional APCs and APC driven proliferation of cells with CD8+ phenotype correlated with the activation of CD8+ T lymphocytes. The responses specific to nucleocapsid protein (NP) were consistently greater than those to the hemagglutinin (HA) at 5 weeks when the CD8+ T cell responses were maximum. By 8 to 9 weeks p.i., responses to either protein were undetectable. The T lymphocytes also responded to stimulation with a heterologous H7N2 AIV infected APCs. Administration of booster dose induced secondary effector cell mediated immune responses which had greater magnitudes than primary effector responses at 10 days p.i. Flow cytometric analysis (FACS) of the T lymphocytes demonstrated that memory CD8+ T lymphocytes of chickens can be distinguished from naive lymphocytes by their higher expression of CD44 and CD45 surface antigens. CD45 expression of memory lymphocytes further increases upon ex vivo stimulation with APCs expressing AIV. This is the first characterization of avian memory responses following both primary and secondary expression of any individual viral protein.

All challenges associated with influenza A viruses including antigenic variation in hemagglutinin (HA) and neuraminidase (NA), the evolving drug resistance and the drawbacks of current vaccines hinder our ability to control this constant threat. Furthermore, gene reassortment as well as the direct transmission of highly pathogenic avian viruses to humans can result in an occasional emergence of novel influenza strains with devastating pandemic potential. Therefore, it is crucial to investigate alternative approaches to better control these viruses and to develop new prophylactic and treatment options. Targeting highly conserved epitopes or antigens among the different subtypes of influenza A virus could offer protection against broad range of influenza viruses, including emerging strains. In my research, I have investigated the potential of broadly neutralizing antibodies against HA and conducted mechanistic study of a prototype vaccine based on the highly conserved nucleoprotein (NP). We recently found that the 14 amino acids of the amino-terminus of the fusion peptide of influenza HA2 subunit is the only universally conserved sequence in all HA subtypes of influenza A and the two lineages of influenza B viruses. Here, I show that universal antibodies targeting this linear sequence in the viral HA (Uni-1 antibodies) can cross-neutralize multiple subtypes of influenza A virus by inhibiting the pH-dependant fusion of viral and cellular membranes. It is noted that the influenza NP is a highly conserved antigen and has the potential to induce heterosubtypic immunity against divergent subtypes of influenza A virus. However, NP-based vaccination only affords weak protective immunity compared to HA. This is mostly due to the non-sterilizing immunity induced by NP. Using CD40 ligand (CD40L), a key regulator of the immune system, as both a targeting ligand and a molecular adjuvant, I show that single immunization with recombinant adenovirus carrying a fused gene encoding the secreted NP-CD40L fusion protein provided robust and long-lasting protection against influenza in normal mice. It enhanced both B-cell and T-cell responses and augmented the role of both NP-specific antibodies and CTLs in protection. Importantly, it afforded effective protection in CD40L and CD4 deficient mice, confirming that the induced protection is CD40L-mediated and CD4+ T cell-independent. The rapid evolution of the influenza A viruses necessitates the development of new alternatives to contain this medically important pathogen. The results of these studies could significantly contribute to future vaccine development and avert the necessity of yearly vaccine updates.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 5 uppsatser.

All challenges associated with influenza A viruses including antigenic variation in hemagglutinin (HA) and neuraminidase (NA), the evolving drug resistance and the drawbacks of current vaccines hinder our ability to control this constant threat. Furthermore, gene reassortment as well as the direct transmission of highly pathogenic avian viruses to humans can result in an occasional emergence of novel influenza strains with devastating pandemic potential. Therefore, it is crucial to investigate alternative approaches to better control these viruses and to develop new prophylactic and treatment options. Targeting highly conserved epitopes or antigens among the different subtypes of influenza A virus could offer protection against broad range of influenza viruses, including emerging strains. In my research, I have investigated the potential of broadly neutralizing antibodies against HA and conducted mechanistic study of a prototype vaccine based on the highly conserved nucleoprotein (NP). We recently found that the 14 amino acids of the amino-terminus of the fusion peptide of influenza HA2 subunit is the only universally conserved sequence in all HA subtypes of influenza A and the two lineages of influenza B viruses. Here, I show that universal antibodies targeting this linear sequence in the viral HA (Uni-1 antibodies) can cross-neutralize multiple subtypes of influenza A virus by inhibiting the pH-dependant fusion of viral and cellular membranes. It is noted that the influenza NP is a highly conserved antigen and has the potential to induce heterosubtypic immunity against divergent subtypes of influenza A virus. However, NP-based vaccination only affords weak protective immunity compared to HA. This is mostly due to the non-sterilizing immunity induced by NP. Using CD40 ligand (CD40L), a key regulator of the immune system, as both a targeting ligand and a molecular adjuvant, I show that single immunization with recombinant adenovirus carrying a fused gene encoding the secreted NP-CD40L fusion protein provided robust and long-lasting protection against influenza in normal mice. It enhanced both B-cell and T-cell responses and augmented the role of both NP-specific antibodies and CTLs in protection. Importantly, it afforded effective protection in CD40L and CD4 deficient mice, confirming that the induced protection is CD40L-mediated and CD4+ T cell-independent. The rapid evolution of the influenza A viruses necessitates the development of new alternatives to contain this medically important pathogen. The results of these studies could significantly contribute to future vaccine development and avert the necessity of yearly vaccine updates.

No description available.

Résumé: Chaque année, les épidémies saisonnières d’influenza causent de 3 à 5 millions de cas sévères de maladie, entraînant entre 250 000 et 500 000 décès mondialement. Seulement deux classes d’antiviraux sont actuellement commercialisées pour traiter cette infection respiratoire : les inhibiteurs de la neuraminidase, tels que l’oseltamivir (Tamiflu) et les inhibiteurs du canal ionique M2 (adamantanes). Toutefois, leur utilisation est limitée par l’apparition rapide de résistance virale. Il est donc d’un grand intérêt de développer de nouvelles stratégies thérapeutiques pour le traitement de l’influenza. Le virus influenza dépend de l’activation de sa protéine de surface hémagglutinine (HA) pour être infectieux. L’activation a lieu par clivage protéolytique au sein d’une séquence d’acides aminés conservée. Ce clivage doit être effectué par une enzyme de l’hôte, étant donné que le génome du virus ne code pour aucune protéase. Pour les virus infectant l’humain, plusieurs études ont montré le potentiel de protéases à sérine transmembranaires de type II (TTSP) à promouvoir la réplication virale : TMPRSS2, TMPRSS4, HAT, MSPL, Desc1 et matriptase, identifiée récemment par notre équipe (Beaulieu, Gravel et al., 2013), activent l’HA des virus influenza A (principalement H1N1 et H3N2). Toutefois, il existe peu d’information sur le clivage de l’HA des virus influenza B, et seulement TMPRSS2 et HAT ont été identifiées comme étant capables d’activer ce type de virus. Les travaux de ce projet de maîtrise visaient à identifier d’autres TTSP pouvant activer l’HA de l’influenza B. L’efficacité de clivage par la matriptase, hepsine, HAT et Desc1 a été étudiée et comparée entre ces TTSP. Ces quatre protéases s’avèrent capables de cliver l’HA de l’influenza B in vitro. Cependant, seul le clivage par matriptase, hepsine et HAT promeut la réplication virale. De plus, ces TTSP peuvent aussi supporter la réplication de virus influenza A. Ainsi, l’utilisation d’un inhibiteur de TTSP, développé en collaboration avec notre laboratoire, permet de bloquer significativement la réplication virale dans les cellules épithéliales bronchiques humaines Calu-3. Cet inhibiteur se lie de façon covalente et lentement réversible au site actif de la TTSP par un mécanisme slow tight-binding. Puisque cet inhibiteur cible une composante de la cellule hôte, et non une protéine virale, il n’entraîne pas le développement de résistance après 15 passages des virus en présence de l’inhibiteur dans les cellules Calu-3. L’inhibition des TTSP activatrices d’HA dans le système respiratoire humain représente donc une nouvelle stratégie thérapeutique pouvant mener au développement d’antiviraux efficaces contre l’influenza. / Abstract: Seasonal influenza epidemics cause between 3 and 5 millions severe cases of disease, leading to 250 000 to 500 000 deaths worldwide. Only two classes of drugs are currently available to treat influenza infections: neuraminidase inhibitors, such as oseltamivir (Tamiflu) and M2 channel inhibitors (adamantanes). However, the use of these antivirals is restricted by rapid emergence of viral resistance. It is therefore of great interest to develop new therapeutic strategies for the treatment of influenza disease. The influenza virus requires activation of its surface protein hemagglutinin (HA) to become infectious. This activation is achieved by proteolytic cleavage in a highly conserved amino acid sequence of the protein. Host cell proteases are responsible for this cleavage since the viral genome doesn’t encode any protease. For viruses that infect humans, many studies have shown the potential of type II transmembrane serine proteases (TTSP) to promote viral replication: TMPRSS2, TMPRSS4, HAT, MSPL, Desc1 and matriptase, recently identified by our team (Beaulieu, Gravel et al., 2013), activate HA of influenza A viruses (mainly H1N1 and H3N2). However, little is known about cleavage of influenza B virus HA, and only TMPRSS2 and HAT have been identified as being capable of activating this type of virus. This project aimed to identify other TTSPs able to activate influenza B HA. Cleavage efficacies of matriptase, hepsin, HAT and Desc1 were studied and compared. These four proteases were shown to be able to cleave influenza B HA using in vitro assays. However, only cleavage by matriptase, hepsin and HAT promoted viral replication. Moreover, these TTSPs also supported the replication of influenza A viruses. Thus, the use of a slow, tight-binding inhibitor (developed in collaboration with our laboratory) that binds to the TTSP active site, forming a covalent and reversible bond, significantly blocked viral replication in human bronchial epithelial Calu-3 cells. Since this inhibitor targets a host cell component, instead of a viral protein, viruses did not develop resistance after 15 passages in presence of the inhibitor in Calu-3 cells. Thus, inhibition of HA-activating TTSPs in the human respiratory tract represents a novel therapeutic strategy against influenza.

Das Hämagglutinin (HA) der Influenzaviren wird während der Assemblierung in Cholesterin- und Sphingolipid-reiche Domänen (Rafts) der Plasmamembran rekrutiert. Vorangehende Studien konnten mittels Fluoreszenzresonanzenergietransfer eine Raft-Integration nachweisen, die von zwei Raft-Zielsignalen abhängig war; zum einen von drei S-acylierten Cysteinen in der zytoplasmatischen Domäne und zum anderen von hydrophoben Aminosäuren (VIL) am Beginn der Transmembrandomäne (TMD). Zudem zeigte sich ein möglicher Einfluss des VIL-Motives auf den intrazellulären Proteintransport. Um diese Annahme zu bestätigen, wurden HA Mutanten in Zellen exprimiert und ihre Ankunft im medialen und trans-Golgi verfolgt. In dieser Arbeit konnte eine Beteiligung des VIL-Motives am Transport bestätigt werden, jedoch nicht der S-Acylierungen. Zudem wurde eine generelle Abhängigkeit des Transportes von der Sphingolipidsynthese beobachtet. Da sowohl die Cholesterinsynthese als auch die Sphingolipidsynthese für den Transport von HA benötigt werden, habe ich die Hypothese aufgestellt, dass das VIL-Motiv in der Lage sein könnte, mit Raft Lipiden zu interagieren. Ein Sequenzvergleich ergab, dass kein Sphingolipid-Bindemotiv vorhanden ist, jedoch ein potenzielles Cholesterin-Consensus-Motiv (CCM, W/Y-I/V/L-K/R). Dieses Motiv wurde nur in der Sequenz von Gruppe 1 jedoch nicht Gruppe 2 HAs gefunden und umfasst das Leucin des VIL Motives. Tatsächlich ist die Mutation des Leucins aber nicht des vorangehenden Isoleucins für den verzögerten Transport verantwortlich. Untersuchungen weiter Einzel- und Mehrfachmutanten konnten eine Abhängigkeit des intrazellulären Transportes von einer möglichen Cholesterinbindung verifizieren. Zudem konnte auch ein zunehmender Effekt auf die Kinetiken vom medialen Golgi zum TGN beobachtet werden, welcher auch die Oberflächenexpression negativ beeinflusste. FLIM-FRET Analysen zeigten zusätzlich eine reduzierte Raft Assoziation der CCMMutanten mit Rafts an der Plasmamembran. Daher kann man spekulieren, dass HA mit Cholesterin interagiert, wodurch sein intrazellulärer Transport durch den Golgi und die Assoziation mit Rafts gewährleistet wird. / During assembly the hemagglutinin (HA) of influenza viruses is recruited to cholesterol- and sphingolipid rich domains of the plasma membrane (lipid rafts). Preceding studies using fluorescence resonance energy transfer showed that lipid-raft integration is dependent on two raft-targeting signals, three S-acylated cysteines located in the cytoplasmic tail and hydrophobic amino acids (VIL) in the part of the transmembrane region (TMR). Furthermore, they gave rise to the assumption that at least the VIL motif might also be important for the intracellular transport of the protein along the exocytic pathway. To verify this assumption, HA mutants were transiently expressed in cells and their arrival in the medial and trans-Golgi compartment was quantified. The observation regarding the involvement of the VIL motif, but not the S-acylation, was verified and a general dependency of HA´s transport on sphingolipid synthesis was detected. Since both cholesterol and sphingolipid synthesis are needed for the transport of HA, I hypothesized that the VIL motif might be able to interact with raft lipids. Sequence alignment revealed no sphingolipid-binding motif, but a putative cholesterol consensus motif (CCM, W/Y-I/V/L-K/R). This CCM is found only in the sequence of group 1 but not group 2 HAs and includes the leucine of the VIL motif. Indeed, mutation of the leucine, but not of the preceding isoleucine is responsible for the delayed transport. Investigation of further single and multiple mutations in the CCM verified a dependency of HA´s intracellular transport on the putative cholesterol-binding motif. Additionally the effect on the kinetics increased from the medial Golgi to the TGN also negatively effecting surface expression. Analysis by FLIM-FRET furthermore displayed a reduced association of HA with mutations in the CCM with lipid rafts at the plasma membrane. Therefore, it is speculated that HA associates with cholesterol, an interaction that facilitates its intracellular transport through the Golgi and association with lipid rafts at the plasma membrane.

Der Viruspartikelzusammenbau hängt von der Anreicherung viraler Untereinheiten in spezifischen Domänen der PM ab. Es wird vorgeschlagen, dass Membran-Rafts – geordnete, sphingomyelin- und cholesterinreiche Mikrodomänen in der PM – als lokale Rekrutierungsstellen dienen. Hämagglutinin (HA) ist ein homotrimeres Glykoprotein in der Hülle des Influenzavirus. Es dient der Bindung an die Wirtszelle und der Fusion mit dem Endosom. Es wird angenommen, dass HA bei der Abschnürung der Viruspartikel von der Zelle mitwirkt. Zwei Hauptbeobachtungen führten zu der Hypothese, dass sich HA in Lipid-Mikrodomänen einlagert: HA wurde biochemisch in Detergens-resistenten Membranen nachgewiesen und die Virushülle ist mit raftbildenden Lipiden angereichert. Um die Rolle der HA-Transmembrandomäne für die Lipid-Raft-Inkorporation aufzuklären, wurde ein Konstrukt entwickelt, das den C-Terminus von HA mit dem gelb fluoreszierenden Protein YFP fusioniert, und die Transmembrandomäne, nicht aber die N-terminale Ektodomäne von HA enthält. In transfizierten Säugetierzellen wurde der Förster-Resonanz-Energie-Transfer (FRET) zwischen diesem Konstrukt und einem GPI-verankerten cyan fluoreszierenden Protein CFP (Raft-Marker) durch Fluoreszenz-Lebenszeit-Mikroskopie (FLIM) gemessen. Die Ergebnisse zeigen, dass sich HA-Konstrukte in Cholesterin-abhängigen Lipiddomänen anreichern, was durch eine erhöhte FRET-Effizienz nachgewiesen wurde. Zudem führen ein Cholesterinentzug aus der PM und die Deletion hochkonservierter Palmitylierungsstellen zu einer signifikanten Verringerung selbiger; sehr gering war diese zwischen dem HA-Konstrukt und einem Nicht-Raft-Marker. Darüberhinaus konnte durch ortsspezifische Mutagenese gezeigt werden, dass die verwendeten Konstrukte disulfidbrückenverbundene Oligomere bilden, welche Voraussetzung für den Transport der Konstrukte an die PM sind. Zeitaufgelöste Anisotropiemessungen ergaben für diese ein starkes Homo-FRET-Signal, welches die Oligomerisierungshypothese bestätigt. / Numerous enveloped viruses bud from the host cell plasma membrane (PM). Assembly of the new viral particles depends on the accumulation of the viral subunits at specific sites of the cell membrane. Lipid domains or rafts enriched of sphingomyelin and cholesterol were suggested as sites for local recruitment of viral components. Hemagglutinin (HA), a homotrimeric glycoprotein embedded in the envelope of influenza virus, mediates binding of the virus to the host cell and fusion between the viral envelope and the endosomal membrane. HA might play an important role in budding of the viral particles from the host cell. Two observations led to the suggestion that HA entraps in lipid microdomains. First, HA was rescued in DRM fractions, second the viral envelope was found to be enriched in lipids generally forming rafts. To elucidate the role of the HA transmembrane domain in lipid raft localization we expressed constructs harboring the transmembrane domain and the cytoplasmic tail but lacking the N-terminal ectodomain of HA in the PM of mammalian cells. We studied energy transfer (FRET) between these constructs and a GPI anchored CFP as a raft marker by fluorescence lifetime imaging microscopy (FLIM). Our results suggest that HA constructs are indeed sorted into cholesterol-dependent lipid domains since cholesterol depletion of the PM caused a significant decrease of FRET efficiency. Likewise, deletion of the three highly conserved palmitoylation sites of HA is also accompanied by a reduction of FRET efficiency. Site directed mutagenesis demonstrated that TMD-HA constructs form disulfide linked oligomers and that oligomerization is fundamental for the transport to the PM. This result was corroborated by time resolved anisotropy measurements that revealed strong homoFRET between TMD-HA-YFP molecules, thus indicating protein clustering. Accordingly, trimerization of full length HA is fundamental for stability and the subsequent delivery of the protein to the cell surface.