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- Member of: ASU Electronic Theses and Dissertations
Description
Random peptide microarrays are a powerful tool for both the treatment and diagnostics of infectious diseases. On the treatment side, selected random peptides on the microarray have either binding or lytic potency against certain pathogens cells, thus they can be synthesized into new antimicrobial agents, denoted as synbodies (synthetic antibodies). On the diagnostic side, serum containing specific infection-related antibodies create unique and distinct "pathogen-immunosignatures" on the random peptide microarray distinct from the healthy control serum, and this different mode of binding can be used as a more precise measurement than traditional ELISA tests. My thesis project is separated into these two parts: the first part falls into the treatment side and the second one focuses on the diagnostic side. My first chapter shows that a substitution amino acid peptide library helps to improve the activity of a recently reported synthetic antimicrobial peptide selected by the random peptide microarray. By substituting one or two amino acids of the original lead peptide, the new substitutes show changed hemolytic effects against mouse red blood cells and changed potency against two pathogens: Staphylococcus aureus and Pseudomonas aeruginosa. Two new substitutes are then combined together to form the synbody, which shows a significantly antimicrobial potency against Staphylococcus aureus (<0.5uM). In the second chapter, I explore the possibility of using the 10K Ver.2 random peptide microarray to monitor the humoral immune response of dengue. Over 2.5 billion people (40% of the world's population) live in dengue transmitting areas. However, currently there is no efficient dengue treatment or vaccine. Here, with limited dengue patient serum samples, we show that the immunosignature has the potential to not only distinguish the dengue infection from non-infected people, but also the primary dengue infection from the secondary dengue infections, dengue infection from West Nile Virus (WNV) infection, and even between different dengue serotypes. By further bioinformatic analysis, we demonstrate that the significant peptides selected to distinguish dengue infected and normal samples may indicate the epitopes responsible for the immune response.
ContributorsWang, Xiao (Author) / Johnston, Stephen Albert (Thesis advisor) / Blattman, Joseph (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2013
Description
The majority of non-small cell lung cancer (NSCLC) patients (70%) are diagnosed with adenocarcinoma versus other histological subtypes. These patients often present with advanced, metastatic disease and frequently relapse after treatment. The tumor suppressor, Liver Kinase B1, is frequently inactivated in adenocarcinomas and loss of function is associated with a highly aggressive, metastatic tumor (1). Identification of the mechanisms deregulated with LKB1 inactivation could yield targeted therapeutic options for adenocarcinoma patients. Re-purposing the immune system to support tumor growth and aid in metastasis has been shown to be a feature in cancer progression (2). Tumor associated macrophages (TAMs) differentiate from monocytes, which are recruited to the tumor microenvironment via secretion of chemotaxic factors by cancer cells. We find that NSCLC cells deficient in LKB1 display increased secretion of C-C motif ligand 2 (CCL2), a chemokine involved in monocyte recruitment. To elucidate the molecular pathway regulating CCL2 up-regulation, we investigated inhibitors of substrates downstream of LKB1 signaling in A549, H23, H2030 and H838 cell lines. Noticeably, BAY-11-7082 (NF-κB inhibitor) reduced CCL2 secretion by an average 92%. We further demonstrate that a CCR2 antagonist and neutralizing CCL2 antibody substantially reduce monocyte migration to NSCLC (H23) cell line conditioned media. Using an in vivo model of NSCLC, we find that LKB1 deleted tumors demonstrate a discernible increase in CCL2 levels compared to normal lung. Moreover, tumors display an increase in the M2:M1 macrophage ratio and increase in tumor associated neutrophil (TAN) infiltrate compared to normal lung. This M2 shift was significantly reduced in mice treated with anti-CCL2 or a CCR2 antagonist and the TAN infiltrate was significantly reduced with the CCR2 antagonist. These data suggest that deregulation of the CCL2/CCR2 signaling axis could play a role in cancer progression in LKB1 deficient tumors.
ContributorsFriel, Jacqueline (Author) / Inge, Landon (Thesis advisor) / Lake, Douglas (Thesis advisor) / Blattman, Joseph (Committee member) / Arizona State University (Publisher)
Created2015
Description
Over the past decade, several high-value proteins have been produced using plant-based transient expression systems. However, these studies exposed some limitations that must be overcome to allow plant expression systems to reach their full potential. These limitations are the low level of recombinant protein accumulation achieved in some cases, and lack of efficient co-expression vectors for the production of multi-protein complexes. This study report that tobacco Extensin (Ext) gene 3' untranslated region (UTR) can be broadly used to enhance recombinant protein expression in plants. Extensin is the hydroxyproline-rich glycoprotein that constitutes the major protein component of cell walls. Using transient expression, it was found that the Ext 3' UTR increases recombinant protein expression up to 13.5- and 6-fold in non-replicating and replicating vector systems, respectively, compared to previously established terminators. Enhanced protein accumulation was correlated with increased mRNA levels associated with reduction in read-through transcription. Regions of Ext 3' UTR essential for maximum gene expression included a poly-purine sequence used as a major poly-adenylation site. Furthermore, modified bean yellow dwarf virus (BeYDV)-based vectors designed to allow co-expression of multiple recombinant genes were constructed and tested for their performance in driving transient expression in plants. Robust co-expression and assembly of heavy and light chains of the anti-Ebola virus monoclonal antibody 6D8, as well as E. coli heat-labile toxin (LT) were achieved with the modified vectors. The simultaneous co-expression of three fluoroproteins using the single replicon, triple cassette is demonstrated by confocal microscopy. In conclusion, this study provides an excellent tool for rapid, cost-effective, large-scale manufacturing of recombinant proteins for use in medicine and industry.
ContributorsRosenthal, Sun Hee (Author) / Mason, Hugh (Thesis advisor) / Mor, Tsafrir (Committee member) / Chang, Yung (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2012
Description
Antibodies are naturally occurring proteins that protect a host during infection through direct neutralization and/or recruitment of the innate immune system. Unfortunately, in some infections, antibodies present unique hurdles that must be overcome for a safer and more efficacious antibody-based therapeutic (e.g., antibody dependent viral enhancement (ADE) and inflammatory pathology). This dissertation describes the utilization of plant expression systems to produce N-glycan specific antibody-based therapeutics for Dengue Virus (DENV) and Chikungunya Virus (CHIKV). The Fc region of an antibody interacts with Fcγ Receptors (FcγRs) on immune cells and components of the innate immune system. Each class of immune cells has a distinct action of neutralization (e.g., antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP)). Therefore, structural alteration of the Fc region results in novel immune pathways of protection. One approach is to modulate the N-glycosylation in the Fc region of the antibody. Of scientific significance, is the plant’s capacity to express human antibodies with homogenous plant and humanized N-glycosylation (WT and GnGn, respectively). This allows to study how specific glycovariants interact with other components of the immune system to clear an infection, producing a tailor-made antibody for distinct diseases. In the first section, plant-produced glycovariants were explored for reduced interactions with specific FcγRs for the overall reduction in ADE for DENV infections. The results demonstrate a reduction in ADE of our plant-produced monoclonal antibodies in in vitro experiments, which led to a greater survival in vivo of immunodeficient mice challenged with lethal doses of DENV and a sub-lethal dose of DENV in ADE conditions. In the second section, plant-produced glycovariants were explored for increased interaction with specific FcγRs to improve ADCC in the treatment of the highly inflammatory CHIKV. The results demonstrate an increase ADCC activity in in vitro experiments and a reduction in CHIKV-associated inflammation in in vivo mouse models. Overall, the significance of this dissertation is that it can provide a treatment for DENV and CHIKV; but equally importantly, give insight to the role of N-glycosylation in antibody effector functions, which has a broader implication for therapeutic development for other viral infections.
ContributorsHurtado, Jonathan (Author) / Chen, Qiang (Thesis advisor) / Arntzen, Charles (Committee member) / Borges, Chad (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2019
Description
Environmental changes are occurring at an unprecedented rate, and these changes will undoubtedly lead to alterations in resource availability for many organisms. To effectively predict the implications of such changes, it is critical to better understand how organisms have adapted to coping with seasonally limited resources. The vast majority of previous work has focused on energy balance as the driver of changes in organismal physiology. While energy is clearly a vital currency, other resources can also be limited and impact physiological functions. Water is essential for life as it is the main constituent of cells, tissues, and organs. Yet, water has received little consideration for its role as a currency that impacts physiological functions. Given the importance of water to most major physiological systems, I investigated how water limitations interact with immune function, metabolism, and reproductive investment, an almost entirely unexplored area. Using multiple species and life stages, I demonstrated that dehydrated animals typically have enhanced innate immunity, regardless of whether the dehydration is a result of seasonal water constraints, water deprivation in the lab, or high physiological demand for water. My work contributed greatly to the understanding of immune function dynamics and lays a foundation for the study of hydration immunology as a component of the burgeoning field of ecoimmunology. While a large portion of my dissertation focused on the interaction between water balance and immune function, there are many other physiological processes that may be impacted by water restrictions. Accordingly, I recently expanded the understanding of how reproductive females can alter metabolic substrates to reallocate internal water during times of water scarcity, an important development in our knowledge of reproductive investments. Overall, by thoroughly evaluating implications and responses to water limitations, my dissertation, when combined previous acquired knowledge on food limitation, will enable scientists to better predict the impacts of future climate change, where, in many regions, rainfall events are forecasted to be less reliable, resulting in more frequent drought.
ContributorsBrusch, George, IV (Author) / DeNardo, Dale F (Thesis advisor) / Blattman, Joseph (Committee member) / French, Susannah (Committee member) / Sabo, John (Committee member) / Taylor, Emily (Committee member) / Arizona State University (Publisher)
Created2019
Description
Despite the safe and effective use of attenuated vaccines for over fifty years, measles virus (MV) remains an insidious threat to global health. Problematically, infants less than one year of age, who are the most prone to severe infection and death by measles, cannot be immunized using current MV vaccines. For this dissertation, I generated and performed preclinical evaluation of two novel MV vaccine candidates. Based on data from clinical trials that showed increasing the dosage of current MV vaccines improved antibody responses in six-month-old recipients, I hypothesized that increasing the relevant antigenic stimulus of a standard titer dose would allow safe and effective immunization at a younger age. I generated two modified MVs with increased expression of the hemagglutinin (H) protein, the most important viral antigen for inducing protective neutralizing immunity, in the background of a current vaccine-equivalent. One virus, MVvac2-H2, expressed higher levels of full-length H, resulting in a three-fold increase in H incorporation into virions, while the second, MVvac2-Hsol, expressed and secreted truncated, soluble H protein to its extracellular environment. The alteration to the virion envelope of MVvac2-H2 conferred upon that virus a measurable resistance to in vitro neutralization. In initial screening in adult mouse models of vaccination, both modified MVs proved more immunogenic than their parental strain in outbred mice, while MVvac2-H2 additionally proved more immunogenic in the gold standard MV-susceptible mouse model. Remarkably, MVvac2-H2 better induced protective immunity in the presence of low levels of artificially introduced passive immunity that mimic the passive maternal immunity that currently limits vaccination of young infants, and that strongly inhibited responses to the current vaccine-equivalent. Finally, I developed a more physiological infant-like mouse model for MV vaccine testing, in which MV-susceptible dams vaccinated with the current vaccine-equivalent transfer passive immunity to their pups. This model will allow additional preclinical evaluation of the performance of MVvac2-H2 in pups of immune dams. Altogether, in this dissertation I identify a promising candidate, MVvac2-H2, for a next generation measles vaccine.
ContributorsJulik, Emily (Author) / Reyes del Valle, Jorge (Thesis advisor) / Chang, Yung (Committee member) / Blattman, Joseph (Committee member) / Hogue, Brenda (Committee member) / Nickerson, Cheryl (Committee member) / Arizona State University (Publisher)
Created2016
Description
Measles is a contagious, vaccine-preventable disease that continues to be the leading
cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins.
Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects.
Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23).
These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker.
cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins.
Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects.
Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23).
These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker.
ContributorsMushtaq, Zuena (Author) / Anderson, Karen (Thesis advisor) / Blattman, Joseph (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2016
Description
The interaction between a virus and its host is a constant competition for supremacy. Both the virus and the host immune system constantly evolve mechanisms to circumvent one another. Vaccinia virus (VACV) infections are a prime example of this. VACV contains a highly conserved innate immune evasion gene, E3L, which encodes the E3 protein composed of a Z-NA-binding domain (Z-NA BD) in the N terminus and a highly characterized dsRNA binding domain in the C-terminus. Both domains of E3 have been found to be essential for the inhibition of antiviral states initiated by host type 1 IFNs. However, the mechanism by which the Z-NA-BD of E3’s N-terminus confers IFN resistance has yet to be established. This is partially due to conflicting evidence showing that the Z-NA-BD is dispensable in most cell culture systems, yet essential for pathogenicity in mice. Recently it has been demonstrated that programmed necrosis is an alternative form of cell death that can be initiated by viral infections as part of the host’s innate immune response to control infection. The work presented here reveals that VACV has developed a mechanism to inhibit programmed necrosis. This inhibition occurs through utilizing E3’s N-terminus to prevent the initiation of programmed necrosis involving the host-encoded cellular proteins RIP3 and Z-NA-binding protein DAI. The inhibition of programmed necrosis has been shown to involve regions of both the viral and host proteins responsible for Z-NA binding through in vivo studies demonstrating that deletions of the Z-NA-BD in E3 correspond to an attenuation of pathogenicity in wild type mice that is restored in RIP3- and DAI-deficient models. Together these findings provide novel insight into the elusive function of the Z-NA-binding domain of the N-terminus and its role in preventing host recognition of viral infections. Furthermore, it is demonstrated that a unique mechanism for resisting virally induced programmed necrosis exists. This mechanism, specific to Z-NA binding, involves the inhibition of a DAI dependent form of programmed necrosis possibly by preventing host recognition of viral infections, and hints at the possible biological role of Z-NA in regulating viral infections.
ContributorsHarrington, Heather (Author) / Jacobs, Bertram L (Thesis advisor) / Langland, Jeffery O (Committee member) / Blattman, Joseph (Committee member) / Haydel, Shelly (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2016
Description
Principle-based ethical frameworks, which commonly make use of codes of ethics, have come to be the popular approach in guiding ethical behavior within scientific research. In this thesis project, I investigate the benefits and shortcomings of this approach, ultimately to argue that codes of ethics are valuable as an exercise in developing a reconciled value profile for a given research community, and also function well as an internal and external proclamation of values and norms. However, this approach results in technical adherence, at best, and given the extent to which scientific research now irreversibly shapes our experience as human beings, I argue for the importance of cultivating ethical virtues in scientific research. In the interest of doing so I explore concepts from Aristotelian virtue ethics, to consider how to ameliorate the shortcomings of principle-based approaches. This project was inspired by a call to research and develop an ethical framework upon which to found a cooperative research network that would be aimed at combating the spread of emerging and re-emerging infectious diseases in resource-restricted countries, specifically throughout Latin America. The desire to found this network on an ethics-based framework is to move beyond technical compliance and cultivate a research community committed to integrity, therefore establishing and maintaining trust and communication that will allow for unprecedented productive collaboration and meaningful outcomes. I demonstrate in this thesis that this requires more than a code of ethics, and use this initiative as a case study to exhibit the merit of integrating concepts from virtue ethics.
ContributorsCraer, Jennifer Ryan (Author) / Ellison, Karin (Thesis advisor) / Sarewitz, Daniel (Committee member) / Blattman, Joseph (Committee member) / Robert, Jason S (Committee member) / Arizona State University (Publisher)
Created2017
Description
Lung metastatic cancers represent a major challenge in both basic and clinical cancer research. The ability to treat lung metastases to date has been challenging, current treatment paradigms are a mix of classic radiotherapy, chemotherapies and tumor-targeted therapies, with no one treatment that is effective for all tumors. Oncolytic viruses (OVs) represent a new therapeutic modality for hard-to-treat tumors. However, major questions still exist in the field, especially around how to therapeutically arm and deliver OVs to sites of disseminated tumors. To address this need, oncolytic myxoma viruses (MYXV) that expresses TNF superfamily member transgenes (vMYX-hTNF or vMyx-mLIGHT) were tested in an immunocompetent syngeneic lung metastatic murine osteosarcoma model. Three versions of this model were used; 1-an early intervention model, 2-an established tumor model, defined by both average tumor burden and failure of anti-PD-L1 and vMyx-TNF monotherapies, and 3-a late-stage disease model, defined by the failure the combination of vMyx-hTNF/PBMCs and anti-PD-L1 therapy. These three models were designed to test different questions about therapeutic efficacy of armed MYXV and delivery of MYXV to lung metastases. In the early intervention model, vMyx-hTNF was found to be an effective therapy, especially when delivered by leukocyte carrier cells (either bone marrow or PBMCs). Next, the combination of immune checkpoint inhibitors, including anti-PD-L1, anti-PD-1 and anti-CTLA-4, with vMyx-TNF/PBMCs were found to increase efficacy in treated mice compared to monotherapies. The established model was used to test potential synergy of vMyx-hTNF with anti-PD-L1 therapy. This model was defined by the failure of the monotherapies, however, in combination, treated mice survived significantly longer, and had lower average tumor burden throughout. This model was also used to test tumor specific delivery using ex vivo loaded PBMCs as carrier cells. Using MYXV expressing Tdtomato, PBMCs were found to deliver MYXV to tumors more effectively than free virus. In the most stringent late-stage disease model, vMyx-mLIGHT/PBMCs and vMyx-mLIGHT/PBMCs plus anti-PD-1 were tested and found to be efficacious where combination vMyx-TNF/PBMCs plus PD-1 failed. These results taken together show that TNFSF arming of MYXV, especially when delivered by autologous PBMCs, represents a new potential treatment strategy for lung metastatic tumors.
ContributorsChristie, John Douglas (Author) / McFadden, Grant (Thesis advisor) / Blattman, Joseph (Committee member) / Jacobs, Bertram (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2021