Network and Algebraic Topology of Influenza Evolution

Evolution is a force that has molded human existence since its divergence from chimpanzees about 5.4 million years ago. In that same amount of time, an influenza virus, which replicates every six hours, would have undergone an equivalent number of generations over only a hundred years. The fast replication times of influenza, coupled with its high mutation rate, make the virus a perfect model to study real-time evolution at a mega-Darwin scale, more than a million times faster than human evolution. While recent developments in high-throughput sequencing provide an optimal opportunity to dissect their genetic evolution, a concurrent growth in computational tools is necessary to analyze the large influx of complex genomic data. In my thesis, I present novel computational methods to examine different aspects of influenza evolution. I first focus on seasonal influenza, particularly the problems that hamper public health initiatives to combat the virus. I introduce two new approaches: 1. The q2-coefficient, a method of quantifying pathogen surveillance, and 2. FluGraph, a technique that employs network topology to track the spread of seasonal influenza around the world. The second chapter of my thesis examines how mutations and reassortment combine to alter the course of influenza evolution towards pandemic formation. I highlight inherent deficiencies in the current phylogenetic paradigm for analyzing evolution and offer a novel methodology based on algebraic topology that comprehensively reconstructs both vertical and horizontal evolutionary events. I apply this method to viruses, with emphasis on influenza, but foresee broader application to cancer cells, bacteria, eukaryotes, and other taxa

Development of an influenza virus vaccine using the baculovirus-insect cell expression system : implications for pandemic preparedness

Key word Influenza, rHA, vaccine, baculovirus, insect cells, production, pandemic preparedness Influenza (or flu) is a highly contagious, acute viral respiratory disease that occurs seasonally in most parts of the world and is caused by influenza viruses. Influenza vaccination is an effective way to reduce the complications and the mortality rate following influenza infections. The currently available influenza vaccines are manufactured in embryonated chicken eggs, a 40-year old production technology. The research in this thesis was aimed at the design, validation and development of a production process for a recombinant hemagglutinin (rHA) influenza vaccine for the prevention of seasonal influenza. The viral surface protein HA is the key antigen in the host response to influenza virus since neutralizing antibodies directed against HA can mitigate or prevent infection. The baculovirus-insect cell system was selected for the synthesis of rHA molecules. The designed process was used to manufacture candidate trivalent rHA vaccines, which were tested in four clinical studies in a total of more than 3000 human subjects age 18 - 92 to support licensure of FluBlok under the “Accelerated Approval” procedure in the United States (U.S.). These studies demonstrated that the purified rHA protein was well tolerated and resulted in a strong and long lasting immune response. In addition, the novel vaccine provided cross protection against drifted influenza viruses. In response to the emergence of the new H1N1 A/California /04/2009 influenza strain, the outlined design was used to produce a rHA vaccine candidate and merely 6 weeks later, the first batches of vaccine were ready for human clinical testing. There are two especially important advantages to the use of this technology from a public health perspective: First, the insect cell-baculovirus system has demonstrated the potential to facilitate safe and expeditious responses to health care emergencies such as the one currently posed by the novel H1N1 virus pandemic and secondly, the rHA vaccine does not contain ovalbumin or other antigenic proteins that are present in eggs and may therefore be administered to people who are egg-allergic. <br/

Investigation of differentially expressed microRNAs in chicken chorioallantoic membrane during influenza A infection

This project involves investigation of differentially regulated microRNAs in chorioallantoic membrane (CAM) of embryonated chicken eggs, which is a significant site of influenza viral replication. First of its kind research in CAM, these findings provide an insight for understanding miRNA-driven gene regulation with a long-term goal of dealing with the global influenza vaccine supply crisis

Development of Inducible Anti-influenza Therapies

Influenza viruses continue to cause significant morbidity and mortality each year despite the development of vaccines and antiviral therapies targeting these viruses. The inherent ability of influenza viruses to accumulate mutations over time has led to the emergence of strains resistant to antiviral therapies. Furthermore, genetic reassortment creates antigenically diverse viruses, making it difficult to develop vaccines that yield broad protection. The objective of the following research studies is to develop two alternative approaches to current methods of antiviral therapeutics.;Six new siRNAs targeting influenza protein expression by RNA interference (RNAi) were characterized. Three siRNAs (M747, M776, M832) knocked down the expression of matrix protein 2 and attenuated influenza infection to a similar degree as MDCK cells treated with a previously published siRNA, M950. The three siRNAs (NS570, NS595, NS615) that target the nonstructural protein 1 and 2 genes promoted the expression of type I interferons, but were unable to attenuate the production of infectious virus. However NS595- and NS615-siRNAs promoted the production of defective interfering viruses. Another siRNA, M331, was able knock down the expression matrix 1 and matrix 2 and attenuate viral replication. Combination siRNA treatment was found to attenuate 20.9% more infectious virus than M950-siRNA treatment alone. Treatment with a single siRNA (M331, NS570, NS595, or NS615) that targets two protein coding sequences was able to knock down the expression of two proteins, thus enhancing the utilities of the siRNAs.;To further take advantage of RNAi as a mechanism to attenuate influenza infection, we developed an inducible anti-influenza therapy containing the influenza conserved promoter that expresses asRNAs only after influenza infection or in the presence of the influenza virus RNA-dependent RNA polymerase (RdRP). asRNA expression was restricted to pM950, pM776, pNS595, or pNA105 treated cells containing the RdRP. The asRNAs expressed from the inducible asRNA expression vectors (pM776 or pNS595) were 84- to 343-fold below the concentration needed to reduce influenza virus infection by RNAi, thus illustrating the need for improved expression kinetics. Limiting expression of asRNAs within influenza infected cells could potentially reduce the adverse effects and limitation of RNAi therapeutics.;In an attempt to reverse antigenic variation and attenuate influenza titer, we developed additional inducible anti-influenza therapies (pUC57 NF-NA and pUC57 F-NA), similar to the inducible asRNA expression vector, which express nonfunctional or functional neuraminidases (NF-NA or F-NA) upon influenza infection. The presence of vector expressed RdRP or influenza infection induced the expression of NF-NA and F-NA. Overexpression of NF-NA was originally hypothesized to attenuate influenza titer; however, NF-NA regained its sialidase activity after RdRP-mediated transcription. pUC57 NF-NA or F-NA transfected cells produced an RNA-intermediate regardless of the presence of the RdRP, whereas the polymerase was required for NF-NA mRNA and protein expression. Interestingly, reinfection of MDCK cells with the supernatant from pUC57 NF-NA or F-NA treated and influenza (N1 subtype) infected cells revealed that the naive MDCK cells generated N2 subtype viruses, indicating the induced N2 viral RNA could be packaged into progeny viruses forcing the N1 virus to become an N2 virus.;These studies demonstrate that RNAi can be an effective means to attenuate influenza infection. Furthermore, incorporation of the influenza conserved promoter into asRNA or neuraminidase expression vectors can be exploited to promote influenza infected cell-specific expression of anti-influenza molecules. This approach may impact the design and advancement of antiviral therapeutics by overcoming the limitations associated with RNAi and allow for current vaccines to protect against influenza infection by forcing influenza viruses to converge into a single subtype

Molecular and clinical insights into seasonal and pandemic influenza

Influenza viruses have caused significant pandemics and epidemics throughout history and continue to be a health problem in humans. New molecular diagnostic assays can be used in the clinical setting to explore relevant clinical manifestations, virus characteristics, and virus epidemiology. This thesis focuses on a variety of subjects related to the molecular diagnosis and clinical consequences of seasonal and pandemic influenza virus infections. Our findings demonstrate that molecular assays are highly sensitive and afford practical detection of resistance mutations, virulence markers and virus expression. We show that computational phylogenetics can provide accurate confirmation of an institutional influenza outbreak. Clinical observational studies reveal that immunocompromised patients can display remarkable prolonged influenza virus excretion with common antiviral resistance development, and that resistant viruses can be transmissible and pathogenic. We demonstrate that host immune responses correlate with virus-associated symptoms and sustained viral clearance. The results described in this thesis confirm that molecular diagnostic assays should be widely implemented in the clinical setting to improve influenza virus laboratory diagnostics. The inherent viral genetic variability and antigenic plasticity is a continuous incentive for new clinical and molecular research to keep up with relevant mutations and to improve our medical understanding of influenza virus infections.UBL - phd migration 201

Mechanisms of resistance to neuraminidase inhibitors in influenza A viruses and evaluation of combined antiviral therapy

Les inhibiteurs de la neuraminidase (INAs) jouent un rôle central dans le contrôle des infections grippales, tant dans le cas des épidémies et des pandémies comme chez les patients immunosuprimés et d'autres patients à risque. Cependant, le développement et la dissémination de la résistance compromettent l'utilité à long terme de cette intervention. En fait, le problème de la résistance aux INAs a été mis en évidence pendant les épidémies de grippe annuelles de 2007-09, avec la dissémination globale d’une variante de la souche A(H1N1) saisonnière résistante à l'oseltamivir. Dans ce cas, les observations préliminaires ont spéculé avec l'existence d'un ensemble de mutations “permissives” qui auraient facilité cette transmission mondiale. Heureusement, l'émergence et la propagation mondiale de la souche pandémique en 2009 a mené au remplacement de la souche saisonnière A/Brisbane/59/2007 (H1N1) résistante à l'oseltamivir, par le virus A(H1N1)pdm09 naturellement sensible aux INA, et, par conséquent, l'oseltamivir a récupéré son utilité clinique. En fait, la plupart des virus A(H1N1)pdm09, A(H3N2) et B circulants à ce jour restent sensibles à l'oseltamivir, avec seulement 1-2% de souches résistantes. Néanmoins, le nombre croissant de souches résistantes récemment détectées en l’absence de traitement fait craindre que ce problème puisse encore augmenter. À cet égard, l'impact de l'émergence et la dissémination de la résistance sur le choix limité des antiviraux actuellement disponibles renforce la nécessité d’une meilleure compréhension des mécanismes sous-jacents à ce phénomène ainsi que de nouvelles approches thérapeutiques. Les différentes études présentées dans le cadre de cette thèse convergent vers l'objectif général de mieux décrire les mécanismes de développement de la résistance aux INAs dans les virus de la grippe. En outre, nous prévoyons que les thérapies combinées pourraient induire une meilleure réponse virologique et immunologique par rapport à la monothérapie antivirale. À la fin, nous nous attendons à ce que notre travail ait un impact sur la gestion des infections grippales en guidant la surveillance mondiale des marqueurs potentiels de résistance, ainsi qu’en proposant des traitements novateurs qui minimisent le développement de souches résistantes.Neuraminidase inhibitors (NAIs) play a central role in the control of influenza infections, with important implications in the management of outbreaks and pandemics as well as in immunocompromised and other at risk patients, with both prophylactic and therapeutic indications. However, the development and dissemination of antiviral drug resistance represents a major limitation that compromises the long-term usefulness of this intervention. Actually, the problem of resistance to NAIs was highlighted by the worldwide dissemination of the oseltamivir-resistant seasonal A(H1N1) neuraminidase H274Y variant during the 2007-09 annual influenza epidemics. In that case, preliminary observations speculated with the existence of a set of “permissive” mutations that could have facilitated this global transmission. Fortunately, the antigenic shift that enabled the emergence of and global spread of the 2009 pandemic strain meant the replacement of the oseltamivir-resistant seasonal A/Brisbane/59/2007 (H1N1) virus by the naturally NAI-susceptible A(H1N1)pdm09 virus, and, consequently, oseltamivir recovered its clinical utility. In fact, most of the circulating A(H1N1)pdm09, A(H3N2) and B viruses remain susceptible to oseltamivir with only 1-2% of tested strains exhibiting phenotypic or genotypic evidence of resistance. Nevertheless, the growing number of resistant strains recently detected in the absence of therapy raises concern that this problem could increase. In that regard, the impact of the emergence and dissemination of resistance on the limited choice of antivirals currently available underscores a better understanding of the mechanisms underlying this phenomenon as well as the necessity for innovative therapeutic approaches. The different studies presented in this thesis converge to the general objective of better describing the mechanisms underlying the development of resistance to NAIs in influenza viruses. Also, we anticipate that combination therapies will induce better virological and immunological responses compared to antiviral monotherapy. In the end, we expect that our work will have an impact on the management of influenza infections by guiding the global surveillance of potential drug resistance markers, as well as proposing innovative ways to improve the clinical outcome and minimizing the development of drug-resistant strains

Applications of large, heterogeneous datasets in understanding and treating pathogenic microbes

Major advances in a myriad of technologies over the past two decades have led to a remarkable increase in the generation of biological data. In response to this increase, researchers have developed methods to pool and analyze large, heterogeneous datasets for novel insights. Here I do just that, leveraging existing data to expand our understanding of therapeutic proteins and pathogenic microbes. In Chapter 2 I outline major shortcomings in existing viral annotation standards using metadata from all influenza A sequences submitted to the GISAID database between 2005 and 2018. I further establish updated nomenclature standards to improve annotation accuracy moving forward. In Chapter 3 I use published Vibrio cholerae sequencing data to derive a comprehensive gene coexpression network. This network provides direct insights into genes influencing pathogenicity, metabolism, and transcriptional regulation, further clarifies results from previous sequencing experiments in V. cholerae, and expands upon micro-array based findings in related gram-negative bacteria. In Chapter 4 I systematically probe all 49,000 unique beta hairpin substructures contained within the Protein Data Bank to uncover key characteristics correlated with stable beta hairpin structure, including amino acid biases and enriched inter-strand contacts. I also establish a set of broad design principles that can be applied to the generation of libraries encoding bioactive proteins. These findings highlight the untapped potential, promise, and power of pooled analyses using large, heterogeneous datasetsCellular and Molecular Biolog

Live attenuated swine influenza vaccine by reverse genetics

Swine influenza (SI) is an acute, highly contagious, respiratory disease of swine. The causative agent of SI infections is swine influenza virus (SIV). SIV is a type A influenza virus classified into the Orthomyxoviridae family and is an enveloped particle with a genome composed of eight negative-orientated RNA segments. The mortality rate of influenza disease in pigs is generally low but morbidity can reach up to 100%. SI infections considerably contribute to respiratory disease in post-weaning pigs, causing significant economic losses due to an increase in the number of days pigs need to reach market weight. In addition, SI infections possess significant human public health concerns. Vaccination is the primary method for the prevention of SI. Currently available vaccines against SI are a combination of two inactivated antigenically distinct SIVs with oil adjuvant. The application of these vaccines induce mainly humoral immune responses. In contrast, application of live attenuated influenza vaccines (LAIV) mimics natural infection and induce strong, long-lived cell-mediated and humoral immunity. Furthermore, LAIV induces cross-protective immunity against different subtypes of influenza A viruses. LAIVs are developed for human and equine influenza viruses but at present no LAIV is available for SIVs. The critical step in influenza virus infection is an initial interaction between virus and cell surface carbohydrates followed by receptor-mediated endocytosis and fusion of the viral and endosomal membranes. Influenza virus entry into cells is mediated by the viral surface glycoprotein hemagglutinin (HA). HA is primary synthesized as a polypeptide in HA0 form. In order to be infectious, HA0 must be cleaved by host proteases into HA1 and HA2 subunits. Therefore, this process is crucial determinant for virus pathogenicity. Our objective was to generate a live attenuated SIVs, particularly a viruses with a modified HA cleavage site resistant to activation during natural infection but which can be activated in vitro by an exogenous protease. Using the reverse genetics technique, we generated two mutant SIVs of strain A/SW/SK/18789/02 (H1N1) containing a modified cleavage site within their HA. Mutant A/SW/SK-R345V (R345V) contained a mutation within HA segment at amino acid (AA) position 345 from Arginine (Arg) to Valine (Val) while the second mutant, A/SW/SK-R345A (R345A) encoded Alanine (Ala) instead of Arginine (Arg) at position AA345 on HA. We showed that HA cleavage in both mutants was strictly dependent on the presence of human neutrophil elastase in tissue culture. These tissue-culture grown mutant SIVs showed similar growth properties in terms of plaque size and growth kinetics, compared to the wild type virus. Both mutant SIVs were able to preserve introduced mutations after multiple passages in tissue culture suggesting that AA substitution within HA cleavage site did not alter genetic stability in the presence of appropriate protease. Furthermore, these mutant SIVs were highly attenuated in pigs but capable of inducing significant cell-mediated and humoral immune responses after two vaccinations via intratracheal (IT) and intranasal (IN) routes. Immune responses induced by vaccination with elastase dependent SIV were sufficient to confer full protection against parental homologous and antigenic variant of H1N1 SIVs and partial protection from heterologous subtypic H3N2 after the challenge. Therefore, elastase-dependent mutant SIV could serve as live vaccine against antigenically distinct swine influenza viruses in pigs

Zoonotic Transmission of Influenza H9 subtype through Reassortment

Influenza A virus causes disease across a broad host range including avian and mammalian species. Most influenza viruses are found in wild aquatic birds, are of low consequence and refrain from zoonotic transmission. However, some strains occasionally cross the species barrier, into domestic birds and a plethora of mammalian species, most notably swine and humans. Many of these infections are dead ends and quickly disappear from the species, but occasionally, a stable lineage is established and becomes endemic in an animal population. Avian Influenza virus (AIV) H9N2 was predominantly found in wild ducks and shore birds across the globe with occasional infections in turkeys until the late 1980's, at which point the virus became established in Eurasian poultry populations. In the late 1990's the virus again jumped hosts, first into swine, and then into humans. Across many regions, these viruses appear to be gaining human-like virus characteristics. Here, the influenza receptor distribution in a range of poultry species has been characterized and shown that many of the birds were able to bind human-like binding viruses. While no large-scale H9N2 human infections have occurred, the threat is there. The most likely route for this to occur is through reassortment with human viruses. The 2009 human pandemic H1N1 (pH1N1) is a likely candidate as it is found in multiple species and seems to readily reassort. The two viruses were shown to be compatible for reassortment and H9:pH1N1 viruses would readily infect and transmit in both ferrets (a human model animal) and swine. Finally, a novel method of modeling reassortment in vivo was developed, which simultaneously tests the breadth of possible reassortant and utilizes natural host selective pressure to select the most-fit progeny. Furthermore, the characterization of these viruses in ferrets showed they readily infect, efficiently transmit, and exhibit mild to moderate pathological consequences. Taken together, these findings broaden our understanding of natural observations, characterize the potential for zoonosis, highlight the dangers H9 viruses may pose to humans, and give scientists a new tool to deepen our understanding of reassortment

Evaluation of the Antiviral Effect of Polyglycerols Functionalized with Sialic Acid on Influenza Virus

Ein vielversprechender Ansatz zur Verhinderung von Infektionen mit Influenzavirus ist die kompetitive Inhibition der Virusanhaftung an die Wirtszellen durch Behinderung der Bindung des viralen Hemagglutinin (HA) an sialylierte Glykanrezeptoren. Allerdings erschwert die hohe Variabilität des HA die Entwicklung von universellen Sialinsäure (SA)-basierten Virostatika. In dieser Arbeit wurde der antivirale Effekt von mit SA funktionalisierten Polyglycerolen (PGs) auf Influenza A Viren (IAV) evaluiert. SA-basierte PGs waren nur bei der Inhibition einer geringen Anzahl an IAV Stämmen effektiv. Um die molekulare Basis für diese Beschränkung zu ergründen, wurden mittels Serienpassagen IAV Mutanten selektiert, die gegen sialyliertes PG resistent waren. Es entwickelten sich drei unabhängige resistente Virusvarianten, die einen einfachen bzw. doppelten Aminosäuren-Austausch in der HA RBS aufwiesen. Durch Hemagglutinations-Elution, Einzel-Virus Kraft-Untersuchungen und Glykanarray Analysen konnte eine verringerte Rezeptorbindungsstabilität sowie ein verändertes Rezeptorbindeprofil für diese Virusvarianten gezeigt werden. Interessanterweise wurden drei unterschiedliche Fälle von Virusbindung und Inhibition beobachtet: 1) Virales HA wurde vom PG gebunden und die Virusreplikation inhibiert, 2) virales HA wurde vom PG gebunden ohne Inhibition der Virusreplikation und 3) Virales HA wurde nicht vom PG gebunden und es gab keine Inhibition. Diese Ergebnisse suggerieren, dass es eine Mindestanforderung an die Affinität oder Avidität für eine effektive kompetitive Inhibition von HA gibt. Durch modifizierte PGs, die Sialyllaktose statt SA und einen Amidlinker enthielten, konnte das Potential von PGs als breite IAV Inhibitoren demonstriert werden. Zusammenfassend bieten die Ergebnisse dieser Arbeit wertvolle Einblicke in die Entwicklung von Resistenzen in IAV gegen Inhibitoren des HA-Attachment und in das strategische Design von sialylierten mutlivalenten Inhibitoren gegen IAV.A promising approach to block influenza virus infections is competitive inhibition of virus attachment to host cells by interfering with binding of the viral surface protein hemagglutinin (HA) to sialylated glycan receptors. However, the high structural and genetic variability of the viral HA has hampered the development of universal sialic acid (SA)-based antivirals. Here, the antiviral effect of biocompatible Polyglycerols (PGs) functionalized with SA on influenza A virus (IAV) was evaluated. PG compounds were only effective at inhibiting a narrow spectrum of IAV strains. To elucidate the molecular basis for this restriction, PG-resistant IAV mutants were selected using serial passaging. Three independent resistant variants developed with single or double amino acid changes mapping to the HA RBS. By employing hemagglutination elution, single-virus force measurements and glycan array analyses, a reduced receptor binding stability as well as an altered receptor binding profile of mutant viruses was shown. Intriguingly, three different cases of virus binding and inhibition were observed using Cy3-labeled compound: 1) viral HA was bound by the compound and resulted in inhibition of replication, 2) viral HA was bound by the compound but replication was not inhibited and 3) viral HA was not bound by the compound and no inhibition occurred. These results suggest that there is an affinity or avidity requirement for effective competitive inhibition of HA attachment. The suitability of PGs as IAV inhibitors with potential for broad activity was demonstrated by a modified PG incorporating sialyllactose instead of SA and an amide linkage covering an extended spectrum of inhibited IAV strains. Taken together, results described in this thesis provide valuable insights into the development of resistance against inhibitors of HA attachment in IAV and into the strategic design of sialylated, multivalent inhibitors aiming at broad activity against influenza viruses