Matching Items (554)
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
ABSTRACT In terms of prevalence, human suffering and costs dengue infections are the most important arthropod-borne viral disease worldwide. Dengue virus (DENV) is a mosquito-borne flavivirus and the etiological agent of dengue fever and dengue hemorrhagic fever. Thus, development of a safe and efficient vaccine constitutes an urgent necessity. Besides the traditional strategies aim at generating immunization options, the usage of viral vectors to deliver antigenic stimulus in order to elicit protection are particularly attractive for the endeavor of a dengue vaccine. The viral vector (MVvac2) is genetically equivalent to the currently used measles vaccine strain Moraten, which adds practicality to my approach. The goal of the present study was to generate a recombinant measles virus expressing structural antigens from two strains of DENV (DENV2 and DENV4) The recombinant vectors replication profile was comparable to that of the parental strain and expresses either membrane bound or soluble forms of DENV2 and DENV4 E glycoproteins. I discuss future experiments in order to demonstrate its immunogenicity in our measles-susceptible mouse model.
ContributorsAbdelgalel, Rowida (Author) / Reyes del Valle, Jorge (Thesis advisor) / Hogue, Brenda (Committee member) / Frasch, Wayne D (Committee member) / Arizona State University (Publisher)
Created2013
Description
Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive ovarian cancer that affects children and young women at a mean age of 24 years. Most SCCOHT patients are diagnosed at an advanced stage and do not respond to chemotherapy. As a result, more than 75% of patients succumb to their disease within 1-2 years. To provide insights into the biological, diagnostic, and therapeutic vulnerabilities of this deadly cancer, a comprehensive characterization of 22 SCCOHT cases and 2 SCCOHT cell lines using microarray and next-generation sequencing technologies was performed. Following histological examination, tumor DNA and RNA were extracted and used for array comparative genomic hybridization and gene expression microarray analyses. In agreement with previous reports, SCCOHT presented consistently diploid profiles with few copy number aberrations. Gene expression analysis showed SCCOHT tumors have a unique gene expression profile unlike that of most common epithelial ovarian carcinomas. Dysregulated cell cycle control, DNA repair, DNA damage-response, nucleosome assembly, neurogenesis and nervous system development were all characteristic of SCCOHT tumors. Sequencing of DNA from SCCOHT patients and cell lines revealed germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (19/24) of SCCOHT patients in addition to SMARCA4 protein loss in 84% (16/19) of SCCOHT tumors, but in only 0.4% (2/485) of other primary ovarian tumors. Ongoing studies are now focusing on identifying treatments for SCCOHT based on therapeutic vulnerabilities conferred by ubiquitous inactivating mutations in SMARCA4 in addition to gene and protein expression data. Our characterization of the molecular landscape of SCCOHT and the breakthrough identification of inactivating SMARCA4 mutations in almost all cases of SCCOHT offers the first significant insight into the molecular pathogenesis of this disease. The loss of SMARCA4 protein is a highly sensitive and specific marker of the disease, highlighting its potential role as a diagnostic marker, and offers the opportunity for genetic testing of family members at risk. Outstanding questions remain about the role of SMARCA4 loss in the biology, histogenesis, diagnosis, and treatment of SCCOHT.
ContributorsRamos, Pilar (Author) / Anderson, Karen (Thesis advisor) / Trent, Jeffrey (Committee member) / Kusumi, Kenro (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2014
Description
Vaccination remains one of the most effective means for preventing infectious diseases. During viral infection, activated CD8 T cells differentiate into cytotoxic effector cells that directly kill infected cells and produce anti-viral cytokines. Further T cell differentiation results in a population of memory CD8 T cells that have the ability to self-renew and rapidly proliferate into effector cells during secondary infections. However during persistent viral infection, T cell differentiation is disrupted due to sustained antigen stimulation resulting in a loss of T cell effector function. Despite the development of vaccines for a wide range of viral diseases, efficacious vaccines for persistent viral infections have been challenging to design. Immunization against virus T cell epitopes has been proposed as an alternative vaccination strategy for persistent viral infections, such as HIV. However, vaccines that selectively engage T cell responses can result in inappropriate immune responses that increase, rather than prevent, disease. Quantitative models of virus infection and immune response were used to investigate how virus and immune system variables influence pathogenic versus protective T cell responses generated during persistent viral infection. It was determined that an intermediate precursor frequency of virus-specific memory CD8 T cells prior to LCMV infection resulted in maximum T cell mediated pathology. Increased pathology was independent of antigen sensitivity or the diversity of TCR in the CD8 T cell response, but was dependent on CD8 T cell production of TNF and the magnitude of initial virus exposure. The threshold for exhaustion of responding CD8 T cells ultimately influences the precursor frequency that causes enhanced disease.In addition, viral infection can occur in the context of co-infection by heterologous pathogens that modulate immune responses and/or disease. Co-infection of two unrelated viruses in their natural host, Ectromelia virus (ECTV) and Lymphocytic Choriomeningitis virus (LCMV) infection in mice, were studied. ECTV infection can be a lethal infection in mice due in part to the blockade of antiviral cytokines, including Type I Interferons (IFN-I). It was determined that ECTV/LCMV co-infection results in decreased ECTV viral load and amelioration of ECTV-induced disease, presumably due to IFN-I induction by LCMV. However, immune responses to LCMV in ECTV co-infected mice were also lower compared to mice infected with LCMV alone and biased toward effector-memory cell generation. Thus, providing evidence for bi-directional effects of viral co-infection that modulate disease and immunity. Together the results suggest heterogeneity in T cell responses during vaccination with viral vectors may be in part due to heterologous virus infection or vaccine usage and that TNF-blockade may be useful for minimizing pathology while maintaining protection during virus infection. Lastly, quantitative mathematical models of virus and T cell immunity can be useful to generate predictions regarding which molecular and cellular pathways mediate T cell protection versus pathology.
ContributorsMcAfee, Megan (Author) / Blattman, Joseph N (Thesis advisor) / Anderson, Karen (Committee member) / Jacobs, Bertram (Committee member) / Hogue, Brenda (Committee member) / Arizona State University (Publisher)
Created2015
Description
Osteosarcoma is the most common bone cancer in children and adolescents. Patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T-lymphocytes (CTL) limit the development of metastatic osteosarcoma. I have investigated the role of Programmed Death Receptor-1 (PD-1) in limiting the efficacy of immune mediated control of metastatic osteosarcoma. I show that human metastatic, but not primary, osteosarcoma tumors express the ligand for PD-1 (PD-L1) and that tumor infiltrating CTL express PD-1, suggesting this pathway may limit CTL control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor infiltrating CTL during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTL in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. My results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy. However, PD-1/PD-L1 blockade treated mice still succumb to disease due to selection of PD-L1 mAb resistant tumor cells via up-regulation of other co-inhibitory T cell receptors. Combinational α-CTLA-4 and α-PD-L1 blockade treated mice were able to completely eradicate metastatic osteosarcoma, and generate immunity to disease. These results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma, although improves survival, may lead to tumor resistance, requiring combinational immunotherapies to combat and eradicate disease.
ContributorsLussier, Danielle (Author) / Blattman, Joseph N. (Thesis advisor) / Anderson, Karen (Committee member) / Goldstein, Elliott (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2015
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
Biological and immunological characterization of plant-produced HIV-1 Gag/dgp41 virus-like particles
Description
Anti-retroviral drugs and AIDS prevention programs have helped to decrease the rate of new HIV-1 infections in some communities, however, a prophylactic vaccine is still needed to control the epidemic world-wide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although recent clinical trials have shown promising results. Recent successes have focused on highly conserved, mucosally-targeted antigens within HIV-1 such as the membrane proximal external region (MPER) of the envelope protein, gp41. MPER has been shown to play critical roles in the viral mucosal transmission, though this peptide is not immunogenic on its own. Gag is a structural protein configuring the enveloped virus particles, and has been suggested to constitute a target of the cellular immunity potentially controlling the viral load. It was hypothesized that HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (dgp41) could be expressed in plants. Plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a tobacco mosaic virus-based expression system or a combination of both. Results of biophysical, biochemical and electron microscopy characterization demonstrated that plant cells could support not only the formation of HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These particles were purified and utilized in mice immunization experiments. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR - a fusion of MPER and the B-subunit of cholera toxin) were administered to BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens could be elicited in mice systemically primed with VLPs and these responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a robust boosting response against Gag and gp41 when boosted with either candidate. Functional assays of these antibodies are in progress to test the antibodies' effectiveness in neutralizing and preventing mucosal transmission of HIV-1. This immunogenicity of plant-based Gag/dgp41 VLPs represents an important milestone on the road towards a broadly-efficacious and inexpensive subunit vaccine against HIV-1.
ContributorsKessans, Sarah (Author) / Mor, Tsafrir S (Thesis advisor) / Matoba, Nobuyuki (Committee member) / Mason, Hugh (Committee member) / Hogue, Brenda (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Description
V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy.
ContributorsWang, Guannan (Author) / Chang, Yung (Thesis advisor) / Levitus, Marcia (Committee member) / Misra, Rajeev (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2012
Description
Plants are a promising upcoming platform for production of vaccine components and other desirable pharmaceutical proteins that can only, at present, be made in living systems. The unique soil microbe Agrobacterium tumefaciens can transfer DNA to plants very efficiently, essentially turning plants into factories capable of producing virtually any gene. While genetically modified bacteria have historically been used for producing useful biopharmaceuticals like human insulin, plants can assemble much more complicated proteins, like human antibodies, that bacterial systems cannot. As plants do not harbor human pathogens, they are also safer alternatives than animal cell cultures. Additionally, plants can be grown very cheaply, in massive quantities.
In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression.
In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression.
ContributorsDiamos, Andy (Author) / Mason, Hugh S (Thesis advisor) / Mor, Tsafrir (Committee member) / Hogue, Brenda (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2017
Description
Breast microcalcifications are a potential indicator of cancerous tumors. Current visualization methods are either uncomfortable or impractical. Impedance measurement studies have been performed, but not in a clinical setting due to a low sensitivity and specificity. We are hoping to overcome this challenge with the development of a highly accurate impedance probe on a biopsy needle. With this technique, microcalcifications and the surrounding tissue could be differentiated in an efficient and comfortable manner than current techniques for biopsy procedures. We have developed and tested a functioning prototype for a biopsy needle using bioimpedance sensors to detect microcalcifications in the human body. In the final prototype a waveform generator sends a sin wave at a relatively low frequency(<1KHz) into the pre-amplifier, which both stabilizes and amplifies the signal. A modified howland bridge is then used to achieve a steady AC current through the electrodes. The voltage difference across the electrodes is then used to calculate the impedance being experienced between the electrodes. In our testing, the microcalcifications we are looking for have a noticeably higher impedance than the surrounding breast tissue, this spike in impedance is used to signal the presence of the calcifications, which are then sampled for examination by radiology.
ContributorsWen, Robert Bobby (Co-author) / Grula, Adam (Co-author) / Vergara, Marvin (Co-author) / Ramkumar, Shreya (Co-author) / Kozicki, Michael (Thesis director) / Ranjani, Kumaran (Committee member) / School of Molecular Sciences (Contributor) / Electrical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05