Ebola virus (EBOV) is an enveloped, negative-sense single-stranded RNA virus that causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes multiple transcripts due to RNA editing at a conserved editing site (ES) (a hepta-uridine stretche). The majority of GP gene transcript is unedited and encodes for a soluble glycoprotein (sGP); a defined function has not been assigned for sGP. In contrast, the transmembrane glycoprotein (GP1,2) dictates viral tropism and is expressed through RNA editing by insertion of a nontemplate adenosine (A) residue. Hypothetically, the insertion/deletion of a different number of A residues through RNA editing would result in another yet unidentified GP gene product, the small soluble glycoprotein (ssGP). I have shown that ssGP specific transcripts were indeed produced during EBOV infection. Detection of ssGP during infection was challenging due to the abundance of sGP over ssGP and the absence of distinguishing antibodies for ssGP. Optimized two- dimensional (2-D) gel electrophoresis verified the expression of ssGP during infection. Biophysical characterization revealed ssGP is a disulfide-linked homodimer that is exclusively N-glycosylated. Although ssGP appears to share similar structural properties with sGP, it does not have the same anti-inflammatory function. Using a new rapid transcript quantification assay (RTQA), I was able to demonstrate that RNA editing is an inherent feature of the genus Ebolavirus and all species of EBOV produce multiple GP gene products. A newly developed dual-reporter minigenome system was utilized to characterize EBOV RNA editing and determined the conserved ES sequence and cis-acting sequences as primary and secondary requirements for RNA editing, respectively. Viral protein (VP) 30, a transcription activator, was identified as a contributing factor of RNA editing— a proposed novel function for this largely uncharacterized viral protein. Finally, I could show that EBOV RNA editing is GP gene-specific because a similar sequence located in L gene did not serve as an ES, most likely due to the lack of the necessary cis-acting sequences. In conclusion, I identified a novel soluble protein of EBOV whose function needs further characterization. I also shed light into the mechanism of EBOV RNA editing, a potential novel target for intervention.

Ebola virus (EBOV) is an enveloped, negative-sense single-stranded RNA virus that causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes multiple transcripts due to RNA editing at a conserved editing site (ES) (a hepta-uridine stretche). The majority of GP gene transcript is unedited and encodes for a soluble glycoprotein (sGP); a defined function has not been assigned for sGP. In contrast, the transmembrane glycoprotein (GP1,2) dictates viral tropism and is expressed through RNA editing by insertion of a nontemplate adenosine (A) residue. Hypothetically, the insertion/deletion of a different number of A residues through RNA editing would result in another yet unidentified GP gene product, the small soluble glycoprotein (ssGP). I have shown that ssGP specific transcripts were indeed produced during EBOV infection. Detection of ssGP during infection was challenging due to the abundance of sGP over ssGP and the absence of distinguishing antibodies for ssGP. Optimized two- dimensional (2-D) gel electrophoresis verified the expression of ssGP during infection. Biophysical characterization revealed ssGP is a disulfide-linked homodimer that is exclusively N-glycosylated. Although ssGP appears to share similar structural properties with sGP, it does not have the same anti-inflammatory function. Using a new rapid transcript quantification assay (RTQA), I was able to demonstrate that RNA editing is an inherent feature of the genus Ebolavirus and all species of EBOV produce multiple GP gene products. A newly developed dual-reporter minigenome system was utilized to characterize EBOV RNA editing and determined the conserved ES sequence and cis-acting sequences as primary and secondary requirements for RNA editing, respectively. Viral protein (VP) 30, a transcription activator, was identified as a contributing factor of RNA editing— a proposed novel function for this largely uncharacterized viral protein. Finally, I could show that EBOV RNA editing is GP gene-specific because a similar sequence located in L gene did not serve as an ES, most likely due to the lack of the necessary cis-acting sequences. In conclusion, I identified a novel soluble protein of EBOV whose function needs further characterization. I also shed light into the mechanism of EBOV RNA editing, a potential novel target for intervention.

Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome by targeting and destroying CD4+ T cells via its Envelope protein (Env), while Ebola virus (EBOV) causes a lethal hemorrhagic fever and targets antigen presenting cells (APCs) via its glycoprotein (GP). There are no licensed vaccines for either virus, posing a problem particularly in Africa, where succumbing to EBOV or HIV is a grim reality. We hypothesized that a replication-competent HIV expressing GP as a replacement for Env will redirect the virus from CD4+ T cells toward antigen presenting cells and act as a live, bivalent vaccine to induce cellular and humoral immune responses against both pathogens, and confer protection against a lethal EBOV challenge in mice. Recombinant HIV-1 molecular clones containing different truncations of the GP gene to replace HIV gp120 were generated and used to rescue three GP-expressing vaccines, HIV-EBOV, HIV-EBOVΔ1, and HIV-EBOVΔ2. These demonstrated tropism for the monocyte cell line, THP-1, and decreased tropism for the CD4+ T cell line, SupT1. While all vaccines induced HIV p24- and GP-specific IFN-γ-secreting T cell responses, HIV-EBOVΔ1 and HIV-EBOVΔ2 induced the most robust responses at 21 days post-vaccination (dpv), respectively. While all vaccines induced total anti-p24 and anti-GP IgG responses, HIV-EBOVΔ1 induced the most robust responses at 42 dpv. HIV-EBOVΔ1 demonstrated the highest protective efficacy against lethal EBOV challenge, followed by HIV-EBOVΔ2 and HIV-EBOV, providing 83%, 67%, and 50% survival in mice, respectively. HIV-EBOVΔ1 shows promise as a protective vaccine against EBOV, but may require further optimization and characterization regarding its mechanism of action and ability to protect against HIV. / October 2016

Thesis supervisor: Wendy J. Maury. Includes bibliographic references (p. 56-66).

SOCIOECONOMIC FACTORS AND THE 2014-16 EBOLA VIRUS DISEASE OUTBREAK IN GUINEA, LIBERIA, AND SIERRA LEONE INTRODUCTION: Ebola virus disease (EVD) is an infectious disease transmitted by close contact with an estimated case fatality rate fluctuating around 50%. The most affected countries by the 2013-16 West African Ebola outbreak were Guinea, Liberia, and Sierra Leone. These countries reported a total of 28616 probable, suspected and confirmed cases. However, we are still learning about the sociodemographic factors that contributed to the outbreak characteristics at the subnational level. METHODS: Data were collected from the World Health Organization, Demographic Health Surveys, and Global Data Lab for 37 districts (8 for Guinea, 15 for Liberia, and 14 for Sierra Leone). The outcome of interest was epidemic size at the district level for Guinea, Liberia, and Sierra Leone (cumulative number of EVD patient confirmed and probable cases). Socio-demographic predictors included household density, sanitation level, mobility, and wealth status. We also controlled for the timing of the start of the outbreak across districts. Pearson’s correlation and multiple linear regression were employed in our analyses. Model building was informed by a review of the relevant literature. Sensitivity analyses were conducted to assess the impact of potential outliers. RESULTS: In the final multivariable regression model, wealth status and household density were positively associated with the epidemic size while sanitation level and the difference in the outbreak start dates were negatively associated with the outcome. These results did not change in the sensitivity analyses. The regression model explained 57% of the variance in epidemic size (Adj R-Sq=0.57), with the largest contribution from the international wealth index (semi-partial R-square=0.22). CONCLUSION: District sociodemographic characteristics such as household density, wealth and sanitation levels contributed to the EVD outbreak in Guinea, Liberia, and Sierra Leone, which is in agreement with recent studies. However, further research should consider other sociodemographic indicators as well as the role of migration and connectivity among regions.

Thesis (M.Sc.)--York University, 2008. Graduate Programme in Chemistry. / Typescript. Includes bibliographical references (leaves 82-88). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38748

Delivery of medicinal products to specific targets can be aided by utilizing different routes of administration. Particular routes may be advantageous when delivering products designed for therapeutic drug delivery, gene therapy, or vaccination. Vaccine candidates must remain stable, be delivered to their proper compartments, and promote sufficient immune responses to their delivered antigens, properties that can be modulated by formulation, adjuvants, and alternate routes of administration. Recently, the nasal passageway has been recognized as a promising route, as mucosally delivered vaccines have the advantage of inducing protection at both mucosal surfaces, a common site of infection, and systemically. Human adenovirus serotype 5 (Ad5) is a candidate vaccine vector capable of being delivered through several routes and inducing strong immune responses to its delivered transgene. The studies presented include vaccination strategies following different routes of administration with various formulation components to determine the ability of Ad5 to deliver its transgene and induce immune responses. The first study screens formulation candidates’ effects on an Ad5-based vaccine’s transduction in vitro, cellular and humoral immune responses in vivo, and efficacy upon challenge in mice. Screening formulation candidates in vitro can eliminate ineffective formulations, thereby limiting animal testing. An Ad5-based Ebola virus vaccine delivered in a combination of mannitol, sucrose, and the surfactant, pluronic F68, improves survival against lethal Ebola challenge in a mouse model compared to delivery in PBS alone. The second study tests the effect of an intravenously delivered Ad5-based vaccine complexed with anti-Ad5 neutralizing antibodies on cellular and humoral immune responses. Different antibody ratios complexed to the Ad5 vector are able to induce disparate cellular and humoral responses. Ratios initiating a strong humoral response towards the Ad5 vector correlate with a reduction of the humoral response against the transgene and few transgene targeted effector T cells. Accordingly, ratios leading to minor humoral responses to the Ad5 vector resulted in stronger humoral responses to the transgene and a strong effector memory T cell response. Taken together, these studies provide insight on how to achieve necessary immune responses in vaccine protocols by testing routes of administration, formulations, and surface modifications of the Ad5 vector. / text

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Ebolavirus is responsible for a deadly hemorrhagic fever that has claimed thousands of lives in Africa and could become a global health threat. Because of the danger of infection, novel Ebola research is restricted to BSL-4 laboratories; this slows progress due to both the cost and expertise required to operate these laboratories. The development of a safe surrogate would speed research and reduce risk to researchers. Two highly conserved Ebola gene segments—from the glycoprotein and nucleoprotein genes—were designed with modifications preventing expression while maintaining sequence integrity, spliced into high copy number plasmids, cloned into E.coli, and tested for stability, safety, and potential research applications. The surrogates were stable over 2-3 months, had a negligible mutation rate (<0.165% over the experiment), and were detectable in human blood down to 5.8E3-1.17E4 surrogates/mL. These protocols could be used to safely simulate other pathogens and promote infectious disease treatment and detection research. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

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