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- Genre: Doctoral Dissertation
- Language: English
Description
Ebola hemorrhagic fever (EHF) is a severe and often fatal disease in human and nonhuman primates, caused by the Ebola virus. Approximately 30 years after the first epidemic, there is no vaccine or therapeutic medication approved to counter the Ebola virus. In this dissertation, a geminiviral replicon system was used to produce Ebola immune complex (EIC) in plant leaves and tested it as an Ebola vaccine. The EIC was produced in Nicotiana benthamiana leaves by fusing Ebola virus glycoprotein (GP1) to the C-terminus of heavy chain of 6D8 monoclonal antibody (mAb), which is specific to the 6D8 epitope of GP1, and co-expressing the fusion with the light chain of 6D8 mAb. EIC was purified by ammonium sulfate precipitation and protein A or protein G affinity chromatography. EIC was shown to be immunogenic in mice, but the level of antibody against Ebola virus was not sufficient to protect the mice from lethal the Ebola challenge. Hence, different adjuvants were tested in order to improve the immunogenicity of the EIC. Among several adjuvants that we used, Poly(I:C), which is a synthetic analog of double-stranded ribonucleic acid that can interact with a Toll-like receptor 3, strongly increased the efficacy of our Ebola vaccine. The mice immunized with EIC co-administered with Poly(I:C) produced high levels of neutralizing anti-Ebola IgG, and 80% of the mice were protected from the lethal Ebola virus challenge. Moreover, the EIC induced a predominant T-helper type 1 (Th1) response, whereas Poly(I:C) co-delivered with the EIC stimulated a mixed Th1/Th2 response. This result suggests that the protection against lethal Ebola challenge requires both Th1 and Th2 responses. In conclusion, this study demonstrated that the plant-produced EIC co-delivered with Poly(I:C) induced strong and protective immune responses to the Ebola virus in mice. These results support plant-produced EIC as a good vaccine candidate against the Ebola virus. It should be pursued further in primate studies, and eventually in clinical trials.
ContributorsPhoolcharoen, Waranyoo (Author) / Mason, Hugh S (Thesis advisor) / Chen, Qiang (Thesis advisor) / Arntzen, Charles J. (Committee member) / Change, Yung (Committee member) / Ma, Julian (Committee member) / Arizona State University (Publisher)
Created2010
Description
Vaccines are one of the most effective ways of combating infectious diseases and developing vaccine platforms that can be used to produce vaccines can greatly assist in combating global public health threats. This dissertation focuses on the development and pre-clinical testing of vaccine platforms that are highly immunogenic, easily modifiable, economically viable to produce, and stable. These criteria are met by the recombinant immune complex (RIC) universal vaccine platform when produced in plants. The RIC platform is modeled after naturally occurring immune complexes that form when an antibody, a component of the immune system that recognizes protein structures or sequences, binds to its specific antigen, a molecule that causes an immune response. In the RIC platform, a well-characterized antibody is linked via its heavy chain, to an antigen tagged with the antibody-specific epitope. The RIC antibody binds to the epitope tags on other RIC molecules and forms highly immunogenic complexes. My research has primarily focused on the optimization of the RIC platform. First, I altered the RIC platform to enable an N-terminal antigenic fusion instead of the previous C-terminal fusion strategy. This allowed the platform to be used with antigens that require an accessible N-terminus. A mouse immunization study with a model antigen showed that the fusion location, either N-terminal or C-terminal, did not impact the immune response. Next, I studied a synergistic response that was seen upon co-delivery of RIC with virus-like particles (VLP) and showed that the synergistic response could be produced with either N-terminal or C-terminal RIC co-delivered with VLP. Since RICs are inherently insoluble due to their ability to form complexes, I also examined ways to increase RIC solubility by characterizing a panel of modified RICs and antibody-fusions. The outcome was the identification of a modified RIC that had increased solubility while retaining high immunogenicity. Finally, I modified the RIC platform to contain multiple antigenic insertion sites and explored the use of bioinformatic tools to guide the design of a broadly protective vaccine.
ContributorsPardhe, Mary (Author) / Mason, Hugh S (Thesis advisor) / Chen, Qiang (Committee member) / Mor, Tsafrir (Committee member) / Wilson, Melissa (Committee member) / Arizona State University (Publisher)
Created2021
Description
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) that emerged from a zoonotic host at the end of 2019 and caused a public health crisis. In this collection of studies, Nicotiana benthamiana plants are used to set the foundation for producing monoclonal antibodies (mAbs) with homogeneous glycosylation to neutralize SARS-CoV-2 and potentially address the immunopathology often observed with severe COVID-19. Specifically, a mAb against the human interleukin (IL)-6 receptor (sarilumab) was generated and evaluated in vitro for its potential to reduce IL-6 signaling that has been shown to be associated with more severe cases of COVID-19. Furthermore, multiple mAbs that bind to the receptor-binding domain (RBD) of SARS-CoV-2 and efficiently neutralize the virus were developed using plant-based expression. Several of these mAbs are from different classes of RBD-binding mAbs that have distinct binding sites from one another. Several mAbs from different classes showed synergy in neutralizing the ancestral strain of SARS-CoV-2 and a smaller subset showed synergy when tested against the highly mutated Omicron (B.1.1.529) variant. Of interest, a novel RBD-binding mAb, termed 11D7, that was raised against the ancestral strain and derived from a hybridoma, appears to have an epitope on the RBD that contributes more synergy to a mAb combination that efficiently neutralizes the B.1.1.529 variant of SARS-CoV-2. This epitope was partially mapped by competitive binding and shows that it overlaps with another known antibody that binds a cryptic, distal epitope, away from the receptor binding site, giving insight into the potential mechanism by which 11D7 neutralizes SARS-CoV-2, as well as potentially allowing it to resist SARS-CoV-2 immune evasion more efficiently. Furthermore, this mAb carries a highly homogeneous glycan pattern when expressed in N. benthamiana, that may contribute to enhanced effector function and provides a tool to elucidate the precise role of crystallizable fragment (Fc)-mediated protection in SARS-CoV-2 infection. Ultimately, these studies provide evidence of the utility of plant-made mAbs to be used as cocktail members, giving clarity to the use of less potent mAbs as valuable cocktail components which will spur further investigations into how mAbs with unique epitopes work together to efficiently neutralize SARS-CoV-2.
ContributorsJugler, Collin (Author) / Chen, Qiang (Thesis advisor) / Lake, Douglas (Committee member) / Steele, Kelly (Committee member) / Mason, Hugh (Committee member) / Arizona State University (Publisher)
Created2022
Description
Scientists are entrusted with developing novel molecular strategies for effective prophylactic and therapeutic interventions. Antivirals are indispensable tools that can be targeted at viral domains directly or at cellular domains indirectly to obstruct viral infections and reduce pathogenicity. Despite their transformative potential in healthcare, to date, antivirals have been clinically approved to treat only 10 out of the greater than 200 known pathogenic human viruses. Additionally, as obligate intracellular parasites, many virus functions are intimately coupled with host cellular processes. As such, the development of a clinically relevant antiviral is challenged by the limited number of clear targets per virus and necessitates an extensive insight into these molecular processes. Compounding this challenge, many viral pathogens have evolved to evade effective antivirals. Therefore, a means to develop virus- or strain-specific antivirals without detailed insight into each idiosyncratic biochemical mechanism may aid in the development of antivirals against a larger swath of pathogens. Such an approach will tremendously benefit from having the specific molecular recognition of viral species as the lowest barrier. Here, I modify a nanobody (anti-green fluorescent protein) that specifically recognizes non-essential epitopes (glycoprotein M-pHluorin chimera) presented on the extra virion surface of a virus (Pseudorabies virus strain 486). The nanobody switches from having no inhibitory properties (tested up to 50 μM) to ∼3 nM IC50 in in vitro infectivity assays using porcine kidney (PK15) cells. The nanobody modifications use highly reliable bioconjugation to a three-dimensional wireframe deoxyribonucleic acid (DNA) origami scaffold. Mechanistic studies suggest that inhibition is mediated by the DNA origami scaffold bound to the virus particle, which obstructs the internalization of the viruses into cells, and that inhibition is enhanced by avidity resulting from multivalent virus and scaffold interactions. The assembled nanostructures demonstrate negligible cytotoxicity (<10 nM) and sufficient stability, further supporting their therapeutic potential. If translatable to other viral species and epitopes, this approach may open a new strategy that leverages existing infrastructures – monoclonal antibody development, phage display, and in vitro evolution - for rapidly developing novel antivirals in vivo.
ContributorsPradhan, Swechchha (Author) / Hariadi, Rizal (Thesis advisor) / Hogue, Ian (Committee member) / Varsani, Arvind (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2022
Description
Coccidioidomycosis, or valley fever (VF), is a fungal infection caused by Coccidioides that is highly endemic in southern Arizona and central California. The antibody response to infection in combination with clinical presentation and radiographic findings are often used to diagnose disease, as a highly sensitive and specific antigen-based assay has yet to be developed and commercialized. In this dissertation, a panel of monoclonal antibodies (mAbs) was generated in an attempt to identify circulating antigen in VF-positive patients. Despite utilizing a mixture of antigens, almost all mAbs obtained were against chitinase 1 (CTS1), a protein previously identified as a main component in serodiagnostic reagents. While CTS1 was undoubtedly a dominant seroreactive antigen, it was not successfully detected in circulation in patient samples prompting a shift toward further understanding the importance of CTS1 in antibody-based diagnostic assays. Interestingly, depletion of this antigen from diagnostic antigen preparations resulted in complete loss of patient IgG reactivity by immunodiffusion. This finding encouraged the development of a rapid, 10-minute point-of-care test in lateral flow assay (LFA) format to exclusively detect anti-CTS1 antibodies from human and non-human animal patients with coccidioidal infection. A CTS1 LFA was developed that demonstrated 92.9% sensitivity and 97.7% specificity when compared to current quantitative serologic assays (complement fixation and immunodiffusion). A commercially available LFA that utilizes a proprietary mixture of antigens was shown to be less sensitive (64.3%) and less specific (79.1%). This result provides evidence that a single antigen can be used to detect antibodies consistently and accurately from patients with VF. The LFA presented here shows promise as a helpful tool to rule-in or rule-out a diagnosis of VF such that patients may avoid unnecessary antibacterial treatments, improving healthcare efficiency.
ContributorsGrill, Francisca J (Author) / Lake, Douglas F (Thesis advisor) / Magee, D Mitch (Committee member) / Grys, Thomas (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2023
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
A roofing manufacturer wants to differentiate themselves from other roofing manufacturers based on performance information. However, construction industry has revealed poor performance documentation in the last couple of decades. With no current developed performance measurement model in the industry, two roofing manufacturers approached the research group to implement a warranty program that measures the performance information of their systems and applicators. Moreover, the success of any project in the construction industry heavily relies upon the capability of the contractor(s) executing the project. Low-performing contractors are correlated with increased cost and delayed schedules, resulting in end-user dissatisfaction with the final product. Hence, the identification and differentiation of the high performing contractors from their competitors is also crucial. The purpose of this study is to identify and describe a new model for measuring manufacturer performance and differentiating contractor performance and capability for two roofing manufacturers (Manufacturer 1 and Manufacturer 2) in the roofing industry. The research uses multiple years of project data and customer satisfaction data collected for two roofing manufacturers for over 1,000 roofing contractors. The performance and end-user satisfaction was obtained for over 7,000 manufacturers' projects and each contractor associated with that project for cost, schedule, and quality metrics. The measurement process was successfully able to provide a performance measurement for the manufacturer based on the customer satisfaction and able to identify low performing contractors. This study presents the research method, the developed measurement model, and proposes a new performance measurement process that entities in the construction industry can use to measure performance.
ContributorsGajjar, Dhaval (Author) / Kashiwagi, Dean (Thesis advisor) / Sullivan, Kenneth (Thesis advisor) / Kashiwagi, Jacob (Committee member) / Arizona State University (Publisher)
Created2016
Description
Saudi Arabia has been facing issues with completing construction projects on time and on budget. It has been documented that 70% of public construction projects are delayed. Studies have identified the low-bid delivery method as an important factor in causing such delays. The procurement system (low-bid) ignores contractors’ performance, and that is reflected in projects’ performance. A case study was performed, at a University campus in northern Saudi Arabia, identifying the major causes of project delays and cost overruns. The University was experiencing delays from 50% to 150%. Also, the actual project costs for four projects were examined and found that all four projects’ costs were higher than the original bid. The delay and cost overruns factors were gathered from the University engineers. A literature research identified one construction management method, best value performance information procurement system (BV PIPS), has documented multiple times its ability to improve project performance. In a comparison using the result of a case study and the results of (BV PIPS), Saudi Arabia’s delivery system was identified as a potential cause of project performance issues. The current procurement system was analyzed and modified to adapt with the (BV PIPS). The proposed procurement system using BV PIPS, which can be implemented in Saudi Arabia, was created with owner side. A large survey was conducted of 761 classified contractors and 43 universities’ representatives who rated causes of delay factors and cost overruns. The delay factors were then compared to delay factors experienced on Saudi construction projects, identified by performing a literature research. The comparison identified 14 important causes of delays. Moreover, the survey showed that classified contractors and universities’ representatives unsatisfied with low-bid, and they agreed with BV PIPS which selecting vendors based on performance with price. The proposed model required a submitted level of experience (LE), risk assessment (RA), and value added (VA). Besides, project managers of vendors should be interviewed during the clarification phase. In addition, venders should submit the project’s scope, technical schedule, milestone schedule, and risk management plan. In the execution phase, vendors should submit a weekly risk report (WRR) and director’s report (DR).
ContributorsAlzara, Majed (Author) / Kashiwagi, Dean (Thesis advisor) / Kashiwagi, Jacob (Committee member) / Al-Tassan, Abdulrahman (Committee member) / Arizona State University (Publisher)
Created2016
Description
Recent studies have identified that contractors in the Saudi construction industry are not the main party that cause risks as owners and other parties have the major share of causing risks. However, with the identification that risks out of contractors’ control are a leading cause of low performance, there is a lack of efficient risk mitigation practices in Saudi to manage these risks. The main aim of this dissertation is to assess the current practices applied by contractors to minimize risk out of their control and develop a risk mitigation model to manage these risks. The main objectives of the study are: investigating the risks that are out of contractors’ control, assessing the contractors’ current risk mitigation and performance measurement practices, and finally developing and validating a risk mitigation model to minimize risks out of contractors’ control and measure performance of involved project parties. To achieve the study aim, a mixed methodological approach was adopted. Theoretical approaches were utilized to review previous research and to develop a conceptual risk mitigation framework followed by a practical approach that is considered with collecting data from contractors. The quantitative method was mainly used to meet the study objectives through distributing a survey in the form of a questionnaire. As a consolidation of the study findings, the top ranked risks that are out of contractors’ control were identified. Furthermore, the results identified that the contractors’ current risk management and performance measurement practices are not effective in minimizing projects risks caused by other parties and ineffective in measuring performance of all parties. The developed model focuses on increasing accountability of project parties through mitigating project parties’ activities and risks with measuring the deviations and identifying sources of deviations. Transparency is utilized in the model through sharing weekly updates of the activities and risks combined with updated information of performance measurements of all project parties. The study results showed that project risks can be minimized and projects’ performance can be increased if contractors shift their focus using the developed model from only managing their own activities and risks to managing all project parties’ activities and risks.
ContributorsAlgahtany, Mohammed (Author) / Sullivan, Kenneth (Thesis advisor) / Kashiwagi, Dean (Committee member) / Badger, William (Committee member) / Arizona State University (Publisher)
Created2018
Description
ABSTRACT
The current Saudi Arabian (SA) procurement system leads to many losses in money and benefits in projects. Also, the use of the traditional procurement system in SA has been identified as one of the causes for poor performance in the delivery of construction and the major risk to the SA government. A questionnaire has been developed and carefully designed based on literature review. The purpose of the survey was to identify the validity of the recent claims that the procurement system in SA is broken and to improve the current SA procurement system. The questionnaire was sent out to 1,396 participants including included 867 engineers, 256 consultants, 93 contractors, 35 owners and 132 architects and 13 academics.
All participants have been registered and licensed professionals at the SA Council for professional engineers, who work in both private and public sectors. The participants are interested in the SA procurement and contracts system with experience ranging from one to more than twenty-five years with the majority of twenty-five years of experience in common construction sectors such as; residential and commercial buildings, healthcare buildings, industrial building and heavy civil construction.
Most of the participants from both private and public sectors agreed with the survey questions subject matter regarding: zone price proposals, contractors' evaluation, risks, planning, projects' scope, owners concern and weekly risks reports (WRR). The survey results showed that the procurement system is the major risk to projects, affects construction projects negatively and is in need of improvement.
Based on the survey and literature review, a model, called Saudi government performance procurement model (SGPPM), has been developed in which the most expert contractor is chosen through four phases: submittals& education, vendors selection, illustration and execution. The resulting model is easy to implement by SA government and does not require special skills or backgrounds.
The current Saudi Arabian (SA) procurement system leads to many losses in money and benefits in projects. Also, the use of the traditional procurement system in SA has been identified as one of the causes for poor performance in the delivery of construction and the major risk to the SA government. A questionnaire has been developed and carefully designed based on literature review. The purpose of the survey was to identify the validity of the recent claims that the procurement system in SA is broken and to improve the current SA procurement system. The questionnaire was sent out to 1,396 participants including included 867 engineers, 256 consultants, 93 contractors, 35 owners and 132 architects and 13 academics.
All participants have been registered and licensed professionals at the SA Council for professional engineers, who work in both private and public sectors. The participants are interested in the SA procurement and contracts system with experience ranging from one to more than twenty-five years with the majority of twenty-five years of experience in common construction sectors such as; residential and commercial buildings, healthcare buildings, industrial building and heavy civil construction.
Most of the participants from both private and public sectors agreed with the survey questions subject matter regarding: zone price proposals, contractors' evaluation, risks, planning, projects' scope, owners concern and weekly risks reports (WRR). The survey results showed that the procurement system is the major risk to projects, affects construction projects negatively and is in need of improvement.
Based on the survey and literature review, a model, called Saudi government performance procurement model (SGPPM), has been developed in which the most expert contractor is chosen through four phases: submittals& education, vendors selection, illustration and execution. The resulting model is easy to implement by SA government and does not require special skills or backgrounds.
ContributorsAlofi, Ahmed Abdulrahman (Author) / Kashiwagi, Dean (Thesis advisor) / Sullivan, Kenneth (Committee member) / Kashiwagi, Jacob (Committee member) / Arizona State University (Publisher)
Created2017