Les virus influenza sont des pathogènes respiratoires responsables d’épidémies saisonnières touchant 10 à 20% de la population mondiale chaque année, constituant donc un problème majeur de santé publique. La vaccination annuelle réduit l’impact des épidémies grippales; cependant, un mésappariement entre les souches vaccinales et circulantes peut parfois survenir et résulter en un échec de protection de la population. Dans ces cas, il est important d’avoir un traitement adéquat afin de traiter l’infection virale. Les inhibiteurs de la neuraminidase (INAs) constituent la principale classe d’antiviraux recommandée pour la prévention et le traitement des infections grippales. Les INAs lient de façon compétitive le site actif de la neuraminidase (NA), ce qui bloque la libération des virions des cellules hôtes inhibant de la sorte la dissémination du virus dans le tractus respiratoire. L’émergence sporadique de virus résistants à l’oseltamivir et/ou au zanamivir avec de faibles taux de transmission a été identifiée lors de traitements des souches saisonnières de l’influenza. Le développement de nouveaux antiviraux devient donc un sujet important d’investigation. Le peramivir, un nouvel INA disponible depuis peu en Amérique du Nord, exerce une activité sur des virus influenza A et B et son efficacité contre des mutants résistants à l’oseltamivir ou au zanamivir n’a pas encore été complètement caractérisée. À cause des différences dans la liaison des INAs avec l’enzyme cible, la nature des mutations de résistance peut varier d’un INA à l’autre bien que certaines mutations pourraient engendrer une résistance croisée à plusieurs INAs. Nous avons démontré que le peramivir s’avère très actif contre les différents sous-types de grippe saisonnière, quoique certains variants aient présentés des phénotypes de multi-résistance à l’oseltamivir, au zanamivir ainsi qu’au peramivir. À cet égard, un nouveau mécanisme de résistance d’un variant menant à la résistance croisée aux INAs a été décrit (I427T/Q313R) dans le cadre de ce mémoire et a permis de comprendre comment des substitutions retrouvées hors du site actif de la NA peuvent affecter la capacité de réplication du virus et sa résistance aux antiviraux. / Influenza viruses are respiratory pathogens responsible for seasonal epidemics affecting 10 to 20% of the world's population every year, thus constituting a major public health impact. Annual vaccination reduces the impact of influenza epidemics; however, a mismatch between the vaccine strain and the circulating strain can sometimes occur and result in an unsuccessful attempt in protecting the population. In such cases, it is important to have adequate treatment to treat influenza infections. Neuraminidase inhibitors (NAIs) are the primary class of antiviral agents recommended for the prevention and treatment of influenza infections. NAIs competitively bind the neuraminidase (NA) active site, blocking the release of virions from host cells and thereby inhibiting the spread of the virus into the respiratory tract. The sporadic emergence of oseltamivir- and/or zanamivir-resistant viruses with low transmission rates was identified in seasonal influenza strains. The development of new antivirals thus became an important subject of investigation. Peramivir, a new NAI recently available in North America, exerts its activity against influenza A and B viruses, but its effectiveness against mutations conferring resistance to oseltamivir or zanamivir has not yet been fully characterized. Due to differences in the binding of NAIs to the target enzyme, the nature of the resistance mutations may vary from one NAI to another, although some mutations could induce global NAI cross-resistance. We have demonstrated that peramivir is highly active against the different seasonal influenza subtypes, although some variants have shown multi-resistance phenotypes to oseltamivir, zanamivir as well as peramivir. In this regard, a new resistance mechanism by which a NA variant leads to NAI cross-resistance (I427T/Q313R) has been described in this thesis and has helped to understand how substitutions found outside the NA active site can affect the replication kinetics of the virus and its resistance to antivirals.

No description available.

Molecular-targeting therapeutics directed towards growth factor receptors have become promising interventions in cancer. They include the family of mammalian receptor tyrosine kinases such as epidermal growth factor, TrkA and insulin. In particular, the insulin receptor (IR) is one of the most well-known members of the RTK family of receptors playing a role in cancer. IRs are covalently-linked heterodimers of αβ subunits on the cell membrane in the absence of insulin. The IR signaling pathways are initially triggered by insulin binding to the α subunits followed by the interaction of β subunits and ATP. The parameter(s) controlling IR activation remains unknown. Here, we report a membrane receptor signaling platform initiated by insulin binding to its receptor to induce Neu1 in live HTC-IR and MiaPaCa-2 cell lines. Microscopy colocalization and co-immunoprecipitation analyses reveal that Neu1 and MMP9 form a complex with naïve and insulin-treated receptors. Tamiflu (neuraminidase inhibitor), galardin and piperazine (broad range MMP inhibitors), MMP9 specific inhibitor and anti-Neu1 antibody blocked Neu1 activity associated with insulin stimulated live cells. Moreover, Tamiflu, anti-Neu1 antibody, and MMP9 specific inhibitor blocked insulin induced insulin receptor substrate-1 phosphorylation (p-IRS1). The previous findings reveal a molecular organizational signaling platform of Neu1 and MMP-9 crosstalk in alliance with insulin receptors. It proposes that insulin binding to the receptor induces MMP9 to activate Neu1, which hydrolyzes α-2,3 sialic acid in removing steric hindrance to generate a functional receptor. The results predict a prerequisite desialylation process by activated Neu1. A complete understanding of IR activation and the role of sialic acids in the iii signaling pathways may provide a therapeutic strategy in the prevention of different diseases such as diabetes mellitus and cancer. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-02-20 11:27:44.861

Influenza A virus (IAV) is one of the most common viruses circulating in the human population and is responsible for seasonal epidemics that affect millions of individuals worldwide. The need to develop new drugs and vaccines against IAVs led scientists to study the main IAV surface antigens hemagglutinin (HA) and neuraminidase (NA). In contrast to HA, which facilitates cell binding and entry of IAVs, NA plays a critical role in the release and spreading of the viral particles. The aim of this thesis was to study how the enzymatic head domain, the stalk and transmembrane domains have evolved to facilitate NA assembly into an enzymatically active homotetramer, and to determine how these regions have evolved together over time. Initially, we observed that the NA transmembrane domain (TMD) assists in the assembly of the head domain by tethering the stalk to the membrane in a tetrameric conformation. Upon examination of the available sequences for NA, we found that the subtype 1 (N1) TMDs have become more polar since 1918 while the subtype 2 (N2) TMDs have consistently retained the expected hydrophobicity of a TMD. Further analysis of the amino-acid sequences revealed a characteristic indicative of an amphipathic assembly for the N1 TMDs that were absent in the TMDs from N2. The function of the amphipathic assembly was examined by creating two viral chimeras, where the original TMD was replaced by another more polar or an engineered hydrophobic TMD. In both cases the viruses carrying the NA TMD chimeras showed reduced growth indicating that the TMD changes created an incompatibility with the head domain of NA. After prolonged passaging of these viruses, natural occurring mutations were observed in the TMD that were able to rescue the defects in viral growth, head domain folding and budding by creating a TMD with the appropriate polar or hydrophobic assembly properties. Interestingly, we observed that N1 and N2 have a great difference in the localization and length of amino-acid deletions occurring in the stalk region. In line with this observation, our data suggests that N1 supports large stalk deletions due to its strong TMD association, whereas N2 requires the presence of a strong oligomerizing stalk region to compensate for its weak TMD interaction. These results have demonstrated how important the NA TMD is for viral infectivity and how the three different domains have evolved in a cooperative manner to promote proper NA assembly / Influensa är en av de mest smittsamma sjukdomarna som drabbar människor och de flesta kan räkna med att bli infekterade många gånger under sin livstid. Influensaviruset attackerar främst luftvägarna, men kan även leda till t.ex. lunginflammation. De enskilda viruspartiklarna (virionerna) kan komma i olika former, men den vanligaste formen som används för att beskriva viruset är den sfäriska. På en virions yta så finns det två olika typer av membranproteiner, som kan liknas med två olika sorters spikar som sticker ut från viruset. Den ena ”spiken” kallas neuraminidas, eller bara kort för NA, och den andra för hemagglutinin (HA). När man har andats in ett influensavirus så kan viruset ta sig till de övre luftvägarna och vidare ner i luftstrupen för att där använda sig av HA för att ta sig in i en cell. Viruset använder sig sedan av cellen för att skapa många nya virioner, som tar sig ut ur cellen för att infektera fler celler. NA är det protein som virionerna använder sig av för att klyva sig loss från modercellen. Målet för avhandlingen var att studera NA och beskriva hur proteinet måste vara ihopsatt för att vara aktivt. NA har en uppbyggnad liknande en trädklunga, där fyra stycken identiska träd (med tillhörande rötter, stammar och trädkronor) går ihop och bildar en enda aktiv enhet, en s.k. tetramer. ”Rötterna” hos NA är den transmembrana domänen (TMD), den del av proteinet som sitter fast i influenaviruskroppen. ”Stammen”, eller stjälkdelen av NA, binder samman TMD med den största delen, huvuddomänen som motsvarar ”trädkronan”. Det är just huvuddomänen som är ansvarig för att klyva loss viruspartiklar från en modercell. Vi har i våra studier sett att det kan vara väldigt viktigt att TMD-domänerna går ihop i grupper om fyra för att hela NA ska kunna gå ihop i en tetramer och aktivt kunna klyva loss viruspartiklarna. När vi studerade TMD från olika influensavirus så märkte vi att vissa egenskaper hos TMD krävs för att de skulle kunna gå ihop, men också att dessa egenskaper inte fanns hos alla influensavirus. Virusen har evolverat över lång tid och har anpassat sig efter värdorganismerna (inklusive människan) och har hittat olika lösningar på problemet med att behöva bilda en tetramer. När vi gjorde ändringar i en TMD som vanligtvis gick ihop till en tetramer, och därmed förhindrade detta, så noterade vi att huvuddomänens funktion påverkades vilket ledde till att influensaviruset hade svårt att spridas. Vidare så har våra pågående studier på stjälkdelen visat att även denna del kan ha stor betydelse för tetrameriseringen av NA, speciellt i de fall där TM-domänen saknar egenskaper för att gå ihop. Avhandlingen tillför inte bara ny och viktig information till influensaforskningen, utan även potentiellt för framställandet av nya influensavacciner/-mediciner. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

No description available.

Abstract not available

The antigenic variability of the Influenza virus hinders our ability to develop new therapeutic and vaccine strategies which provide a broad protection against all influenza strains. It has been previously suggested that a means to approach this challenge is to identify conserved sequences within viral proteins and use these for future therapeutic targets. Although such conserved sequences are plentiful amongst the internal viral proteins, their lack of exposure to the host immune system makes mounting an immune response against these regions difficult. Alternatively, the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) have been shown to provide host protection against a limited number of influenza strains when used as vaccine targets; however conserved regions within these proteins which are also antibody accessible are extremely rare. My Ph.D. thesis project is focused on investigating the functional role of a conserved region within the NA protein and to further determine the protection afforded by a monoclonal antibody to this region. In a comprehensive bioinformatics analysis, the only universally conserved sequence amongst all influenza A and B viral NA has been previously identified as being located between amino acids (a.a.) 222-230 (dubbed the HCA-2 region). However, the potential role of this region remains largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics and growth kinetics, I have found that substitutions in this sequence significantly affect viral replication by impairing the catalytic activity, substrate-binding and thermostability of NA. These findings prompted me to further investigate if antibody to this region may provide protection against influenza infection. Indeed, universal monoclonal antibody (HCA-2 MAb) against this peptide provided broad inhibition against all nine subtypes of NA in vitro and heterosubtypic protection in mice challenged with lethal doses of mouse-adapted viruses. I further demonstrated that residues within this peptide that are exposed on the surface of NA and located in close proximity to the active site, I222 and E227, are indispensable for antibody-mediated inhibition. These data are the first to demonstrate a monoclonal antibody against the NA protein which provides heterosubtypic protection. Since I observed that the HCA-2 antibody provided a broad inhibition against all nine subtypes of influenza A NA, I decided to investigate whether this inhibitory effect could be extended against Influenza B. Here, I have further reported that HCA-2 MAb provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. I also demonstrate that the growth and NA enzymatic activity of two drug resistant influenza B strains are also inhibited by the HCA-2 antibody. The findings of my Ph.D. thesis project have thus demonstrated that the HCA-2 region is paramount to optimal viral function. Additionally, my data show that antibodies generated against this region provide heterosubtypic protection both in vitro and in vivo and against drug resistant strains. These results indicate that this universally conserved epitope should be further explored as a potential target for future antiviral intervention and vaccine-induced immune responses.

Thesis (Ph.D.) - University of St Andrews, May 2009.

Techn. Hochsch., Diss., 2002--Aachen.

The antigenic variability of the Influenza virus hinders our ability to develop new therapeutic and vaccine strategies which provide a broad protection against all influenza strains. It has been previously suggested that a means to approach this challenge is to identify conserved sequences within viral proteins and use these for future therapeutic targets. Although such conserved sequences are plentiful amongst the internal viral proteins, their lack of exposure to the host immune system makes mounting an immune response against these regions difficult. Alternatively, the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) have been shown to provide host protection against a limited number of influenza strains when used as vaccine targets; however conserved regions within these proteins which are also antibody accessible are extremely rare. My Ph.D. thesis project is focused on investigating the functional role of a conserved region within the NA protein and to further determine the protection afforded by a monoclonal antibody to this region. In a comprehensive bioinformatics analysis, the only universally conserved sequence amongst all influenza A and B viral NA has been previously identified as being located between amino acids (a.a.) 222-230 (dubbed the HCA-2 region). However, the potential role of this region remains largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics and growth kinetics, I have found that substitutions in this sequence significantly affect viral replication by impairing the catalytic activity, substrate-binding and thermostability of NA. These findings prompted me to further investigate if antibody to this region may provide protection against influenza infection. Indeed, universal monoclonal antibody (HCA-2 MAb) against this peptide provided broad inhibition against all nine subtypes of NA in vitro and heterosubtypic protection in mice challenged with lethal doses of mouse-adapted viruses. I further demonstrated that residues within this peptide that are exposed on the surface of NA and located in close proximity to the active site, I222 and E227, are indispensable for antibody-mediated inhibition. These data are the first to demonstrate a monoclonal antibody against the NA protein which provides heterosubtypic protection. Since I observed that the HCA-2 antibody provided a broad inhibition against all nine subtypes of influenza A NA, I decided to investigate whether this inhibitory effect could be extended against Influenza B. Here, I have further reported that HCA-2 MAb provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. I also demonstrate that the growth and NA enzymatic activity of two drug resistant influenza B strains are also inhibited by the HCA-2 antibody. The findings of my Ph.D. thesis project have thus demonstrated that the HCA-2 region is paramount to optimal viral function. Additionally, my data show that antibodies generated against this region provide heterosubtypic protection both in vitro and in vivo and against drug resistant strains. These results indicate that this universally conserved epitope should be further explored as a potential target for future antiviral intervention and vaccine-induced immune responses.