Stalking Flu: Development and Characterization of a Broadly Neutralizing Monoclonal Antibody Targeting the Influenza Hemagglutinin Stem

Seasonal epidemics caused by influenza A viruses (IAV) result in an estimated 290,000- 650,000 deaths worldwide each year (17). While antivirals targeted to influenza exist, resistance to these drugs is increasing and regular vaccination remains the most effective way to prevent infection (26, 73, 99). However due to the persistence of antigenic drift and shift, influenza vaccines must be updated each season and antigenic mismatches can reduce efficacy (24, 118). Immunity to influenza either from vaccination or infection is principally mediated by antibodies generated to one of its major surface proteins, Hemagglutinin (HA). HA is a homotrimer, each monomer HA0 is composed of a globular head and stem (100, 111). Proteases present in the host cleave native HA0 into two subunits, HA1 and HA2. Residues in HA1 form the receptor binding pocket within the head and facilitate interaction with the host cell. The stem is encoded by both HA1 and HA2 and contains the fusion peptide required for cytosolic release (10, 112). Most antibodies isolated from patients following infection or vaccination are targeted to the receptor binding domain of the HA head. These antibodies are efficient neutralizers but highly strain specific, recognizing only antigenically similar strains of the same IAV group and consequently lose relevance rapidly as viruses undergo drift (68, 141). However, patients immunized with heterosubtypic HA can also develop antibodies that target the HA stem domain. These antibodies are often effective against multiple HA subtypes, reflecting strong structural constraints imposed on the HA stem epitope, and are referred to as “broadly neutralizing”. It is because of this strong conservation that there has been an interest in characterizing HA stem antibodies in the development of a universal flu vaccine (22, 115, 123). Since their discovery in 1991, dozens xi of broadly neutralizing stem antibodies have been isolated in humans and even more have been derived using targeted technologies, including hybridoma cell generation (69). We used two immunization protocols that each featured successive injection of influenza with antigenically distinct HA components, H1 then H3, followed by hybridoma cell development to generate candidate monoclonal antibodies (mAbs) targeted to the IAV surface glycoproteins. We screened antibody containing hybridoma supernatant for activity against influenza proteins using ELISA, Neuraminidase Inhibition (NI), and Hemagglutinin Inhibition (HAI) assays. Those antibodies which showed appreciable activity against the immunizing viruses from hybridoma generation were purified for further study. Of 114 antibodies, mAb 1G3 (IgG1) was found by Western blotting to successfully bind the HA protein of H1, H1pdm, and H3 influenza A viruses as well as influenza B viruses of both major lineages (Yamagata and Victoria). Significantly, mAb 1G3 was able to successfully neutralize both influenza A and B viruses in vitro as shown by plaque assay and indirect immunofluorescence of infected cells. 1G3 was also shown to be effective against H1, H3, and an influenza B virus in ovo, delaying onset of positive viral titer and preventing viral expansion overall. Although we believe mAb 1G3 to have a conformational epitope, linear epitope mapping revealed that 1G3 likely binds in the stalk domain near to the fusion peptide and across the C terminus of HA1 and N terminus of HA2. We found this locus to be strongly conserved among heterosubtypic influenza strains, including influenza B. Importantly, only one other antibody known as CR9114 has been identified that can neutralize both influenza A and B viruses in vivo (32). CR9114’s epitope appears to have significant overlap with the proposed epitope of our mAb 1G3, although they are not identical. Where CR9114 xii displayed no activity against B viruses in vitro, 1G3 is the only antibody to date that inhibits both A and B viruses in cells. This previously unseen neutralization profile may suggest the possible presence of an additional inhibition mechanism or mechanisms active in cell culture. While CR9114 prevents the fusogenic conformational change of HA within the host endosome and incites Antibody Dependent Cellular Cytotoxicity (ADCC) in vivo, we have demonstrated through Western blotting and neuraminidase inhibition that our mAb 1G3 likely also prevents extracellular HA cleavage and interferes with virion budding (11, 29, 122). Therefore, mAb 1G3 is a unique and previously uncharacterized antibody that is broadly neutralizing against Group 1 and Group 2 influenza A as well as both lineages of influenza B

Time to Raise the Bar: The Real Corporate Social Responsibility Report for the Hershey Company

This document is part of a digital collection provided by the Martin P. Catherwood Library, ILR School, Cornell University, pertaining to the effects of globalization on the workplace worldwide. Special emphasis is placed on labor rights, working conditions, labor market changes, and union organizing.ILRF_Time_to_Raise_the_Bar_Hershey.pdf: 2396 downloads, before Oct. 1, 2020

A Survey to Determine the Basis for Junior High School Girls’ Physical Education Report Card Grades

It was the purpose of this study (1) to examine the grading practices of junior high school girls’ physical education teachers and to question selected ninth grade students and their parents concerning the grade or mark on the report card; and (2) to develop a system of report card marking for girls’ physical education which can be recommended on the basis of these findings

The behaviour of children one year after a head injury

Bibliography: leaves 117-128.This study examines the behavioural sequelae in 116 subjects aged between 5 and 14 years 1 year after a head injury. Subjects were selected from consecutive admissions to trauma units on the basis of neurological criteria. Severity of injury was graded according to duration of post-traumatic amnesia (PTA). There were 53 children in the Moderate group with PTA less than 24 hours; 37 children in the Severe group with PTA between 1 and 7 days and 26 children in the Very Severe group with PTA over 7 days. The head-injured subjects were matched for age, sex, ethnic group and socio-economic background with 37 Controls who had trauma not involving the head. There are two main objectives to this thesis. The first is to establish which of the behaviours present in the head-injured children at one year follow-up could be attributed to their head injury. This was achieved in two ways: By separating behaviours which presented for the first time in the post-traumatic period from those with a pre-traumatic origin and through the identification of other factors such as pre-morbid behaviour patterns and persistent psychosocial adversity which may have contributed to post-head injury behaviour patterns. The above two steps made it possible to determine the existence of a defined post-traumatic syndrome. The second main objective was simply to establish whether there was a dose-response relationship between the severity of the head injury and behavioural sequelae. The areas examined included physical complaints, developmental problems, activity levels, social problems, disturbances of mood and control, neurotic behaviour and mental symptoms

Patch Life: Army Wives Behind the Wire

In 2014, one hundred and eighty-four female civilian women moved overseas with the British Army for a tour lasting three years. Known by the collective ‘Army Wives’ and ‘Dependents,’ these women faced multiple dislocations as they sought to make a life for themselves, bounded by British military jurisdiction in a South-Eastern corner of the European Union. Using a free association qualitative interview methodology, twenty-nine women married to serving soldiers and officers of the British Army were interviewed during a six-month fieldwork period at an overseas British military garrison. In addition to their individual narratives, this thesis draws on participant observation and field diary extracts and, based on a range of sociological and feminist theories, it reveals these women’s negotiation of, and reflections upon, their time as incorporated overseas army wives. Attuned to the wives’ voices, Patch Life, Army Wives Behind the Wire accompanies them as they learn the complex unwritten rules of the overseas garrison, becoming physically and ontologically bounded into the hierarchical and patriarchal system of their husbands’ employer. It explores how women become incorporated and institutionalised, investigating how they negotiate and experience their associated positioning within and parallel to the ranked boundaries of the overseas military garrison. It considers what shapes and governs and influences their incorporation (or not) into their military marriage. This thesis probes how these women (re)create their identity within the socio-spaces of the garrison through the bonds of friendship and community living in a geographically remote location. Lying at the heart of this research is how these civilian women respond to their socially constructed appellation of ‘army wife’, whether they embraced its traditions and expectations or, if in their refutation, they were able to find an alternative sense of self located for three years overseas living a patch life behind the wire

The Disruptive Power of Frame Narratives in Mary Shelley\u27s KEEPSAKE Tales

Thanks to the success of Frankenstein, scholars have begun to analyze one of Mary Shelley’s signature techniques, the frame narrative, within her other novels. However, scholarly discourse on the subject routinely leaves out one aspect of Shelley’s oeuvre that has received relatively little attention in general — her short stories for The Keepsake (1828-1857). In this essay, I propose that Shelley deliberately uses frame narratives in her short stories to both illuminate and deconstruct the structural, temporal and ideological constraints imposed upon her by publishing within the periodical space. Drawing on narratological and feminist readings of Shelley’s work, I illustrate how two of Shelley’s framed tales, “The Sisters of Albano” (1829) and “The Swiss Peasant” (1831), subtly call into question the “cult of beauty” and Romantic aestheticization of the “ordinary” within The Keepsake and other literary annuals. This argument thus fills a critical void in Shelley studies by properly contextualizing her tales within their own framing device, the literary annual itself

Evolutionary History of a Large-Genome Salamander Across Five Disjunct Regions of the Southeastern United States

The southeastern United States has a dynamic geologic history. Mountain range uplift, sea level fluctuations, and river basin evolution have shaped species geographic distributions in the region. My dissertation objectively identifies amphibian species with highly fragmented distributions in the Southeast (Chapter 2) and examines the phylogeographic history of one of those species, Plethodon serratus. This salamander species has a widely disjunct range across five regions: the Appalachians, the Ozarks, the Ouachitas, and two allopatric sites in Louisiana. Analyses of mitochondrial and nuclear loci (Chapter 3) showed that P. serratus is comprised of multiple genetic lineages, and the five regions are not reciprocally monophyletic. Instead, there was evidence of historical gene flow between one of the Louisiana sites and the Ouachitas. Niche and paleodistribution modeling results suggested that P. serratus expanded from the Appalachians during the cooler Last Glacial Maximum and has since been restricted to its current disjunct distribution by a warming climate. These data reject the universal applicability of the glacial contraction model to temperate taxa and reiterate the importance of considering the natural history of individual species. Using a large next-generation sequencing data set of ultraconserved elements (Chapter 4), I estimated a fully resolved species tree that confirmed the non-sister relationship of the two Louisiana populations. I successfully generated this data set without modifying the established laboratory protocols; this is an important note, as large genomes can present challenges for next-generation sequencing. I found that P. serratus has a genome size of 21 pg (Chapter 5) and that this species, along with its closest relatives, has undergone genome size reduction since diverging from the ancestor of Plethodon. This dissertation demonstrates that established methods of next-generation sequencing can be used in studies of large-genome salamander phylogeography. This is especially important, as many salamander populations are declining in the face of habitat loss and climate change. Plethodon serratus is only one of many amphibian species in the Southeast with fragmented ranges. Future research on the biogeography of these organisms will be crucial to conservation

Discriminating customers, responsible patients, empowered users: consumerism and the modernisation of health care

The centrality of patient choice in the recent political rhetoric of both New Labour and the Conservative Party has prompted a renewed interest in the shift towards a more consumerist conception of health care in the UK. Accordingly, this article reports on early findings from a project in the ESRC/AHRB Cultures of Consumption Programme, exploring how the 'consumer' is constituted in narratives of health reform, and the ways in which policy documents present a particular image of the consumer as a rationale for institutional and cultural change. The article then goes on to look at the ways in which service delivery organisations have responded to New Labour's consumerist imperative. Drawing upon a series of interviews with senior health care managers in two case study locations, the article highlights ways in which choice, responsibility and empowerment have become critical points at which a consumerist orientation is articulated with established professional cultures, and how health organisations have experienced – and attempted to resolve – the tensions that result