Matching Items (18)
Description
Vaccinia virus (VACV) is the current vaccine for the highly infectious smallpox disease. Since the eradication of smallpox, VACV has been developed extensively as a heterologous vaccine vector for several pathogens. However, due to the complications associated with this replication competent virus, the safety and efficacy of VACV vaccine vector has been reevaluated. To evaluate the safety and efficacy of VACV, we study the interactions between VACV and the host innate immune system, especially the type I interferon (IFN) signaling pathways. In this work, we evaluated the role of protein kinase R (PKR) and Adenosine Deaminase Acting on RNA 1(ADAR1), which are induced by IFN, in VACV infection. We found that PKR is necessary but is not sufficient to activate interferon regulatory factor 3 (IRF3) in the induction of type I IFN; and the activation of the stress-activated protein kinase/ c-Jun NH2-terminal kinase is required for the PKR-dependent activation of IRF3 during VACV infection. Even though PKR was found to have an antiviral effect in VACV, ADAR1 was found to have a pro-viral effect by destabilizing double stranded RNA (dsRNA), rescuing VACVΔE3L, VACV deleted of the virulence factor E3L, when provided in trans. With the lessons we learned from VACV and host cells interaction, we have developed and evaluated a safe replication-competent VACV vaccine vector for HIV. Our preliminary results indicate that our VACV vaccine vector can still induce the IFN pathway while maintaining the ability to replicate and to express the HIV antigen efficiently. This suggests that this VACV vector can be used as a safe and efficient vaccine vector for HIV.
ContributorsHuynh, Trung Phuoc (Author) / Jacobs, Bertram L (Thesis advisor) / Hogue, Brenda (Committee member) / Chang, Yung (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2013
Description
The concept of vaccination dates back further than Edward Jenner's first vaccine using cowpox pustules to confer immunity against smallpox in 1796. Nevertheless, it was Jenner's success that gave vaccines their name and made vaccinia virus (VACV) of particular interest. More than 200 years later there is still the need to understand vaccination from vaccine design to prediction of vaccine efficacy using mathematical models. Post-exposure vaccination with VACV has been suggested to be effective if administered within four days of smallpox exposure although this has not been definitively studied in humans. The first and second chapters analyze post-exposure prophylaxis of VACV in an animal model using v50ΔB13RMγ, a recombinant VACV expressing murine interferon gamma (IFN-γ) also known as type II IFN. While untreated animals infected with wild type VACV die by 10 days post-infection (dpi), animals treated with v50ΔB13RMγ 1 dpi had decreased morbidity and 100% survival. Despite these differences, the viral load was similar in both groups suggesting that v50ΔB13RMγ acts as an immunoregulator rather than as an antiviral. One of the main characteristics of VACV is its resistance to type I IFN, an effect primarily mediated by the E3L protein, which has a Z-DNA binding domain and a double-stranded RNA (dsRNA) binding domain. In the third chapter a VACV that independently expresses both domains of E3L was engineered and compared to wild type in cells in culture. The dual expression virus was unable to replicate in the JC murine cell line where both domains are needed together for replication. Moreover, phosphorylation of the dsRNA dependent protein kinase (PKR) was observed at late times post-infection which indicates that both domains need to be linked together in order to block the IFN response. Because smallpox has already been eradicated, the utility of mathematical modeling as a tool for predicting disease spread and vaccine efficacy was explored in the last chapter using dengue as a disease model. Current modeling approaches were reviewed and the 2000-2001 dengue outbreak in a Peruvian region was analyzed. This last section highlights the importance of interdisciplinary collaboration and how it benefits research on infectious diseases.
ContributorsHolechek, Susan A (Author) / Jacobs, Bertram L (Thesis advisor) / Castillo-Chavez, Carlos (Committee member) / Frasch, Wayne (Committee member) / Hogue, Brenda (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2011
Description
HIV/AIDS is the sixth leading cause of death worldwide and the leading cause of death among women of reproductive age living in low-income countries. Clinicians in industrialized nations monitor the efficacy of antiretroviral drugs and HIV disease progression with the HIV-1 viral load assay, which measures the copy number of HIV-1 RNA in blood. However, viral load assays are not widely available in sub-Saharan Africa and cost between 50-$139 USD per test on average where available. To address this problem, a mixed-methods approach was undertaken to design a novel and inexpensive viral load diagnostic for HIV-1 and to evaluate barriers to its adoption in a developing country. The assay was produced based on loop-mediated isothermal amplification (LAMP). Blood samples from twenty-one individuals were spiked with varying concentrations of HIV-1 RNA to evaluate the sensitivity and specificity of LAMP. Under isothermal conditions, LAMP was performed with an initial reverse-transcription step (RT-LAMP) and primers designed for HIV-1 subtype C. Each reaction generated up to a few billion copies of target DNA within an hour. Presence of target was detected through naked-eye observation of a fluorescent indicator and verified by DNA gel electrophoresis and real-time fluorescence. The assay successfully detected the presence of HIV in samples with a broad range of HIV RNA concentration, from over 120,000 copies/reaction to 120 copies/reaction. In order to better understand barriers to adoption of LAMP in developing countries, a feasibility study was undertaken in Tanzania, a low-income country facing significant problems in healthcare. Medical professionals in Northern Tanzania were surveyed for feedback regarding perspectives of current HIV assays, patient treatment strategies, availability of treatment, treatment priorities, HIV transmission, and barriers to adoption of the HIV-1 LAMP assay. The majority of medical providers surveyed indicated that the proposed LAMP assay is too expensive for their patient populations. Significant gender differences were observed in response to some survey questions. Female medical providers were more likely to cite stigma as a source problem of the HIV epidemic than male medical providers while males were more likely to cite lack of education as a source problem than female medical providers.
ContributorsSalamone, Damien Thomas (Author) / Jacobs, Bertram L (Thesis advisor) / Marsiglia, Flavio (Committee member) / Stout, Valerie (Committee member) / Johnson, Crista (Committee member) / Arizona State University (Publisher)
Created2011
Description
Coronaviruses are a medically significant group of viruses that cause respiratory and enteric infections in humans and a broad range of animals. Coronaviruses assemble at the internal membranes of the endoplasmic reticulum- Golgi intermediate compartment (ERGIC). While there is a basic understanding of how viruses assemble at these membranes, the full mechanistic details are not understood. The coronavirus envelope (E) protein is a small multifunctional viroporin protein that plays a role in virus assembly but its function is unknown. The two goals of this study were : 1. To identify and analyze the localization of MHV E and 2. To identify the functions of conserved residues in the tail of the E protein. This study closely examined the localization, dynamics and mobility of the mouse hepatitis virus (MHV) E protein to gain insight into its functions. The results from the first aim of this study showed that the MHV E protein localizes at the site of assembly in the ERGIC-Golgi region based on analysis by immunofluorescence and correlative electron microscopy. A novel tetra-cysteine tagged MHV E protein was used to study the dynamics of the protein in cells. A recombinant MHV E Lumio virus was used to study the trafficking and mobility of the E protein. Live cell imaging and surface biotinylation confirmed that the E protein does not traffic to the cell surface. Fluorescence recovery after photo-bleaching (FRAP) analyses revealed that the E protein is mobile at the site of localization. As a part of the second aim, conserved prolines and tyrosine in the tail of the protein were targeted by site directed mutagenesis and analyzed for functionality. While none of the residues were absolutely essential for localization or virus production, the mutations had varying degrees of effect on envelope formation, protein stability and virus release. Differential scanning calorimetry data suggests that the proline and tyrosine residues enhance interaction with lipids. A wild type (WT) peptide contained the conserved residues was also able to significantly reduce the hexagonal phase transition temperature of lipids, whereas a mutant peptide with alanine substitutions for the residues did not cause a temperature shift. This suggests that the peptide can induce a negative curvature in lipids. The E protein may be playing a role as a scaffold to allow membrane bending to initiate budding or possibly scission. This data, along with the localization data, suggests that the E protein plays a mechanistic role at the site of virus assembly possibly by remodeling the membrane thereby allowing virus budding and/or scission.
ContributorsVenkatagopalan, Pavithra (Author) / Hogue, Brenda G (Thesis advisor) / Jacobs, Bertram L (Committee member) / Roberson, Robert W. (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2012
Description
CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self
onself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy.
onself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy.
ContributorsKrishna, Sri (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Committee member) / Jacobs, Bertram L (Committee member) / Lake, Douglas F (Committee member) / Arizona State University (Publisher)
Created2017
Description
Cell death is a powerful tool through which organisms can inhibit the spread of viruses by preventing their replication. In this work, I used viral and chemical stressors to elucidate the mechanisms by which one anti-viral system might be activated over another, focusing on the programmable death pathway necroptosis and Protein Kinase R (PKR). PKR can detect viral dsRNA and trigger antiviral effects such as cessation of translation and induction of programmed death. Necroptosis is a rapid cellular death that can be induced via sensors such as DNA-dependent activator of IFN-regulatory factors (DAI), also known as Z-DNA-binding protein 1 (ZBP1). DAI contains a Z-form nucleic acid (ZNA) binding domain. E3, the primary vaccinia virus (VACV) interferon resistance protein, contains a similar domain in its amino terminus. We have previously reported this domain to be necessary for the inhibition of both PKR activation and DAI/ZBP1-mediated necroptosis.
Monkeypox virus is a reemerging human pathogen. Despite a partial amino-terminal deletion in its E3 homolog, it does not activate PKR. In chapter 2, I show that MPXV produces less dsRNA than VACV, which could explain how the virus avoids activating PKR.
The amino-terminus of vaccinia is associated with ZNA binding, inhibition of PKR, and inhibition of necroptosis. To determine the roles of PKR inhibition and ZNA binding in necroptosis inhibition, I characterized the VACV mutants Za(ADAR1)-E3, which binds ZNA but does not inhibit PKR, and E3:Y48A, which cannot bind ZNA. I found that while Za(ADAR1)-E3 fails to induce necroptosis, E3:Y48A does not activate PKR but does induce necroptosis. This suggests that Z-form nucleic acid binding is not necessary for vaccinia E3-mediated inhibition of PKR, nor is the inhibition of PKR sufficient for the inhibition of necroptosis.
Finally, all known ZNA-binding proteins have immune functions and home to stress granules. I asked if stress granule formation alone could lead to necroptosis. I found that in L929 cells sodium arsenite, a known inducer of stress granules, could trigger DAI-dependent necroptosis. This suggests that DAI/ZBP1 is not necessarily a sensor of viral ligands but perhaps is a sensor of stress signals brought about by infection.
Monkeypox virus is a reemerging human pathogen. Despite a partial amino-terminal deletion in its E3 homolog, it does not activate PKR. In chapter 2, I show that MPXV produces less dsRNA than VACV, which could explain how the virus avoids activating PKR.
The amino-terminus of vaccinia is associated with ZNA binding, inhibition of PKR, and inhibition of necroptosis. To determine the roles of PKR inhibition and ZNA binding in necroptosis inhibition, I characterized the VACV mutants Za(ADAR1)-E3, which binds ZNA but does not inhibit PKR, and E3:Y48A, which cannot bind ZNA. I found that while Za(ADAR1)-E3 fails to induce necroptosis, E3:Y48A does not activate PKR but does induce necroptosis. This suggests that Z-form nucleic acid binding is not necessary for vaccinia E3-mediated inhibition of PKR, nor is the inhibition of PKR sufficient for the inhibition of necroptosis.
Finally, all known ZNA-binding proteins have immune functions and home to stress granules. I asked if stress granule formation alone could lead to necroptosis. I found that in L929 cells sodium arsenite, a known inducer of stress granules, could trigger DAI-dependent necroptosis. This suggests that DAI/ZBP1 is not necessarily a sensor of viral ligands but perhaps is a sensor of stress signals brought about by infection.
ContributorsJohnson, Brian Patrick (Author) / Jacobs, Bertram L (Thesis advisor) / Blattman, Joseph N (Committee member) / Langland, Jeffrey O (Committee member) / Stout, Valerie G (Committee member) / Arizona State University (Publisher)
Created2018
Description
The distinguishing feature of the filamentous fungi is the hyphae - tube-like microscopic cells that exhibit polarized growth via apical extension and allow the fungus to interact with its environment. Fungi elongate at the hyphal apex, through the localized construction of new plasma membrane and cell wall through the exocytosis of secretory vesicles. One population of these vesicles have been identified as chitosomes, containing chitin synthase isoenzymes, which are responsible for the polymerization of N-acetylglucosamine from UDP N-acetylglucosamine into chitin, the primary fibrillar component of the fungal cell wall. The chitosomes, in addition to other vesicles, can be observed aggregating in the hyphal tip in most filamentous fungi. In the Ascomycota and Basidiomycota, this collection of vesicles exhibits discrete organization and has been termed a Spitzenkörper. Although accumulations of vesicles can be observed in the hyphal tip of many growing filamentous fungi, some debate continues as to what precisely defines a Spitzenkörper. This study reports the details of three separate projects: first, to document the effects of deleting a single chitin synthase, CHS-1 and CHS-6 in Neurospora crassa with regards to hyphal ultrastructure, cytoplasmic organization, and growth in comparison to the wild-type. Given the importance of chitin synthesis in fungal cell growth, deletion of a critical chitin synthase presumably impacts cell wall structure, fungal growth and cytoplasmic organization. Second, an examination of the ultrastructure of four zygomycetous fungi - Coemansia reversa, Mortierella verticillata, Mucor indicus, and Gilbertella persicaria has been conducted. Utilization of cryofixation and freeze-substitution techniques for electron microscopy has produced improved preservation of cytoplasmic ultrastructure, particularly at the hyphal apex, allowing detailed analysis of vesicle size, contents, and organization. Lastly, hyphal tip organization was reviewed in a broad range of fungi. Previous studies had either focused on a few select fungi or representative groups. Vesicle organization, composition and size do appear to vary among the classes of fungi, but some trends, like the vesicle crescent in the zygomycetous fungi have been documented.
ContributorsFisher, Karen Elizabeth (Author) / Roberson, Robert W. (Thesis advisor) / Chandler, Douglas (Committee member) / Riquelme, Meritxell (Committee member) / Stutz, Jeam (Committee member) / Wojciechowski, Martin (Committee member) / Arizona State University (Publisher)
Created2015
Description
The remarkable conservation of molecular and intra-/inter-cellular pathways underpinning the fundamental aspects of sugar partitioning in two evolutionarily divergent organisms – a non-vascular moss Physcomitrella patens and a vascular cereal crop Oryza sativa (rice) – forms the basis of this manuscript. Much of our current knowledge pertaining to sugar partitioning in plants mainly comes from studies in thale cress, Arabidopsis thaliana, but how photosynthetic sugar is loaded into the phloem in a crop as important as rice is still debated. Even less is known about the mechanistic aspects of sugar movement in mosses. In plants, sugar either moves passively via intercellular channels called plasmodesmata, or through the cell wall spaces in an energy-consuming process. As such, I first investigated the structure of plasmodesmata in rice leaf minor vein using electron tomography to create as of yet unreported 3D models of these channels in both simple and branched conformations. Contrary to generally held belief, I report two different 3D morphotypes of simple plasmodesmata in rice. Furthermore, the complementary body of evidence in arabidopsis implicates plasma membrane localized Proton Pyrophosphatase (H+-PPase) in the energy-dependent movement of sugar. Within this wider purview, I studied the in situ ultrastructural localization patterns of H+-PPase orthologs in high-pressure frozen tissues of rice and physcomitrella. Were H+-PPases neo-functionalized in the vascular tissues of higher plants? Or are there evolutionarily conserved roles of this protein that transcend the phylogenetic diversity of land plants? I show that H+-PPases are distinctly expressed in the actively growing regions of both rice and physcomitrella. As expected, H+-PPases were also localized in the vascular tissues of rice. But surprisingly, H+-PPase orthologs were also prominently expressed at the gametophyte-sporophyte junction of physcomitrella. Upon immunogold labeling, H+-PPases were found to be predominantly localized at the plasma membrane of the phloem complexes of rice source leaves, and both the vacuoles and plasma membrane of the transfer cells in the physcomitrella haustorium, linking H+-PPases in active sucrose loading in both plants. As such, these findings suggest that the localization and presumably the function of H+-PPases are conserved throughout the evolutionary history of land plants.
ContributorsRegmi, Kamesh Chandra (Author) / Gaxiola, Roberto A (Thesis advisor) / Wojciechowski, Martin F (Committee member) / Pigg, Kathleen B (Committee member) / Roberson, Robert W. (Committee member) / Arizona State University (Publisher)
Created2016
Evolutionary analysis of the CAP superfamily of proteins using amino acid sequences and splice sites
Description
Here I document the breadth of the CAP (Cysteine-RIch Secretory Proteins (CRISP), Antigen 5 (Ag5), and the Pathogenesis-Related 1 (PR)) protein superfamily and trace some of the major events in the evolution of this family with particular focus on vertebrate CRISP proteins. Specifically, I sought to study the origin of these CAP subfamilies using both amino acid sequence data and gene structure data, more precisely the positions of exon/intron borders within their genes. Counter to current scientific understanding, I find that the wide variety of CAP subfamilies present in mammals, where they were originally discovered and characterized, have distinct homologues in the invertebrate phyla contrary to the common assumption that these are vertebrate protein subfamilies. In addition, I document the fact that primitive eukaryotic CAP genes contained only one exon, likely inherited from prokaryotic SCP-domain containing genes which were, by nature, free of introns. As evolution progressed, an increasing number of introns were inserted into CAP genes, reaching 2 to 5 in the invertebrate world, and 5 to 15 in the vertebrate world. Lastly, phylogenetic relationships between these proteins appear to be traceable not only by amino acid sequence homology but also by preservation of exon number and exon borders within their genes.
ContributorsAbraham, Anup (Author) / Chandler, Douglas E. (Thesis advisor) / Buetow, Kenneth H. (Committee member) / Roberson, Robert W. (Committee member) / Arizona State University (Publisher)
Created2016
Description
The HIV-1 pandemic continues to cause millions of new infections and AIDS-related deaths each year, and a majority of these occur in regions of the world with limited access to antiretroviral therapy. Therefore, an HIV-1 vaccine is still desperately needed. The most successful HIV-1 clinical trial to date used a non-replicating canarypox viral vector and protein boosting, yet its modest efficacy left room for improvement. Efforts to derive novel vectors which can be both safe and immunogenic, have spawned a new era of live, viral vectors. One such vaccinia virus vector, NYVAC-KC, was specifically designed to replicate in humans and had several immune modulators deleted to improve immunogenicity and reduce pathogenicity. Two NYVAC-KC vectors were generated: one expressing the Gag capsid, and one with deconstructed-gp41 (dgp41), which contains an important neutralizing antibody target, the membrane proximal external region (MPER). These vectors were combined with HIV-1 Gag/dgp41 virus-like particles (VLPs) produced in the tobacco-relative Nicotiana benthamiana. Different plant expression vectors were compared in an effort to improve yield. A Geminivirus-based vector was shown to increase the amount of MPER present in VLPs, thus potentially enhancing immunogenicity. Furthermore, these VLPs were shown to interact with the innate immune system through Toll-like receptor (TLR) signaling, which activated antigen presenting cells to induce a Th2-biased response in a TLR-dependent manner. Furthermore, expression of Gag and dgp41 in NYVAC-KC vectors resulted in activation of antiviral signaling pathways reliant on TBK1/IRF3, which necessitated the use of higher doses in mice to match the immunogenicity of wild-type viral vectors. VLPs and NYVAC-KC vectors were tested in mice, ultimately showing that the best antibody and Gag-specific T cell responses were generated when both components were administered simultaneously. Thus, plant-produced VLPs and poxvirus vectors represent a highly immunogenic HIV-1 vaccine candidate that warrants further study.
ContributorsMeador, Lydia Rebecca (Author) / Mor, Tsafrir S (Thesis advisor) / Jacobs, Bertram L (Thesis advisor) / Blattman, Joseph N (Committee member) / Mason, Hugh S (Committee member) / Arizona State University (Publisher)
Created2016