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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

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
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Description
Infections caused by the Hepatitis C Virus (HCV) are very common worldwide, affecting up to 3% of the population. Chronic infection of HCV may develop into liver cirrhosis and liver cancer which is among the top five of the most common cancers. Therefore, vaccines against HCV are under intense study

Infections caused by the Hepatitis C Virus (HCV) are very common worldwide, affecting up to 3% of the population. Chronic infection of HCV may develop into liver cirrhosis and liver cancer which is among the top five of the most common cancers. Therefore, vaccines against HCV are under intense study in order to prevent HCV from harming people's health. The envelope protein 2 (E2) of HCV is thought to be a promising vaccine candidate because it can directly bind to a human cell receptor and plays a role in viral entry. However, the E2 protein production in cells is inefficient due to its complicated matured structure. Folding of E2 in the endoplasmic reticulum (ER) is often error-prone, resulting in production of aggregates and misfolded proteins. These incorrect forms of E2 are not functional because they are not able to bind to human cells and stimulate antibody response to inhibit this binding. This study is aimed to overcome the difficulties of HCV E2 production in plant system. Protein folding in the ER requires great assistance from molecular chaperones. Thus, in this study, two molecular chaperones in the ER, calreticulin and calnexin, were transiently overexpressed in plant leaves in order to facilitate E2 folding and production. Both of them showed benefits in increasing the yield of E2 and improving the quality of E2. In addition, poorly folded E2 accumulated in the ER may cause stress in the ER and trigger transcriptional activation of ER molecular chaperones. Therefore, a transcription factor involved in this pathway, named bZIP60, was also overexpressed in plant leaves, aiming at up-regulating a major family of molecular chaperones called BiP to assist protein folding. However, our results showed that BiP mRNA levels were not up-regulated by bZIP60, but they increased in response to E2 expression. The Western blot analysis also showed that overexpression of bZIP60 had a small effect on promoting E2 folding. Overall, this study suggested that increasing the level of specific ER molecular chaperones was an effective way to promote HCV E2 protein production and maturation.
ContributorsHong, Fan (Author) / Mason, Hugh (Thesis advisor) / Gaxiola, Roberto (Committee member) / Chang, Yung (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2011
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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

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
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Description
This study focused on the connection between the EnvZ/OmpR two-component regulatory system and the iron homeostasis system in Escherichia coli, specifically how a mutant form of EnvZ11/OmpR is able to reduce the expression of fepA::lacZ, a reporter gene fusion in E. coli. FepA is one of several outer membrane siderophore

This study focused on the connection between the EnvZ/OmpR two-component regulatory system and the iron homeostasis system in Escherichia coli, specifically how a mutant form of EnvZ11/OmpR is able to reduce the expression of fepA::lacZ, a reporter gene fusion in E. coli. FepA is one of several outer membrane siderophore receptors that allow extracellular siderophores bound to iron to enter the cells to power various biological processes. Previous studies have shown that in E. coli cells that expressed a mutant allele of envZ, called envZ11, which led to altered expression of various iron genes including down regulation of fepA::lacZ. The wild type EnvZ/OmpR system is not considered to regulate iron genes, but because these envz11 strains had downregulated fepA::lacZ, this study was undertaken to understand the connection and mechanisms of this downregulation. A large number of Lac+ revertants were obtained from the B32-2483 strain (envz11 and fepA::lacZ) and 7 Lac+ revertants that had reversion mutations not directly correcting the envZ11 allele were further characterized. With P1 phage transduction genetic mapping that involved moving a kanamycin resistance marker linked to fepA::lacZ, two Lac+ revertants were found to have their reversion mutations in the fepA promoter region, while the other five revertants had their mutations mapping outside the fepA region. These two revertants underwent DNA sequencing and found to carry two different single base pair mutations in two different locations of the fepA promoter region. Each one is in the Fur repressor binding region, but one also may have affected the Shine-Dalgarno region involved in translation initiation. All 7 reveratants underwent beta-galactosidase assays to measure fepA::lacZ expression. The two revertants that had mutations in the fepA promoter region had significantly increased fepA activity, with the revertant with the Shine-Dalgarno mutation having the most elevated fepA expression. The other 5 revertants that did not map in the fepA region had fepA expression elevated to the same level as that found in the wild type EnvZ/OmpR background. The data suggest that the negative effect of envZ11 can be overcome by multiple mechanisms, including directly correcting the envZ11 allele or changing the fepA promoter region.
ContributorsKalinkin, Victor Arkady (Co-author) / Misra, Rajeev (Co-author, Thesis director) / Mason, Hugh (Committee member) / Foy, Joseph (Committee member) / Biomedical Informatics Program (Contributor) / School of Life Sciences (Contributor) / W. P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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Description
Overexpression of AVP1 (Arabidopsis vacuolar pyrophosphatase), a type I H+ pyrophosphatase, results in greater biomass, possibly due to a function in sucrose transport within the phloem. Overexpression of the phloem lipid-associated family protein (PLAFP) was shown to increase the number of vascular bundles in Arabidopsis. Could these two phenotypes complement

Overexpression of AVP1 (Arabidopsis vacuolar pyrophosphatase), a type I H+ pyrophosphatase, results in greater biomass, possibly due to a function in sucrose transport within the phloem. Overexpression of the phloem lipid-associated family protein (PLAFP) was shown to increase the number of vascular bundles in Arabidopsis. Could these two phenotypes complement one another additively? In this work, double mutants overexpressing both AVP1 and PLAFP were characterized. These double mutants have enhanced biomass, greater leaf area, and a larger number of vascular bundles than the single mutant lines. Overexpression of PLAFP does not result in any increase in rhizosphere acidification capacity.
ContributorsWilson, Sean (Co-author) / Furstenau, Tara (Co-author) / Gaxiola, Roberto (Thesis director) / Mason, Hugh (Committee member) / Wojciechowski, Martin (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05
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Description
The Intercellular Adhesion Molecule-1 (ICAM-1, known as CD54) is a cell surface type I transmembrane glycoprotein with a molecular weight of 85 to 110 kDa. The primary function of ICAM-1 is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 is used as a receptor for

The Intercellular Adhesion Molecule-1 (ICAM-1, known as CD54) is a cell surface type I transmembrane glycoprotein with a molecular weight of 85 to 110 kDa. The primary function of ICAM-1 is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 is used as a receptor for various pathogens such as rhinoviruses, coxsackievirus A21 and the malaria parasite Plasmodium falciparum. ICAM-1 contains five immunoglobulin (Ig) domains in its long N-terminal extracellular region, a hydrophobic transmembrane domain, and a small C-terminal cytoplasmic domain. The Ig domains 1-2 and Ig domains 3-4-5 have been crystallized separately and their structure solved, however the full ICAM-1 structure has not been solved. Because ICAM-1 appears to be important for the mediation of cell-to-cell communication in physiological and pathological conditions, gaining a structural understanding of the full-length membrane anchored ICAM-1 is desirable. In this context, we have transiently expressed a plant-optimized gene encoding human ICAM-1 in Nicotiana benthamiana plants using the MagnICON expression system. The plant produced ICAM-1 is forming aggregates according to previous data. Thus, the current extraction and purification protocols have been altered to include TCEP, a reducing agent. The protein was purified using TALON metal affinity resin and partially characterized using various biochemical techniques. Our results show that there is a reduction in aggregation formation with the use of TCEP.
ContributorsPatel, Heeral (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Kannan, Latha (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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Description
Variants of human butyrylcholinesterase (BChE) have been designed to have high cocaine hydrolytic activity. These variants have potential pharmacological applications toward treating cocaine overdose and addiction. These enzymes must be stable in the human body over fairly long periods of time in order to be effective at treating cocaine addiction.

Variants of human butyrylcholinesterase (BChE) have been designed to have high cocaine hydrolytic activity. These variants have potential pharmacological applications toward treating cocaine overdose and addiction. These enzymes must be stable in the human body over fairly long periods of time in order to be effective at treating cocaine addiction. Recombinantly expressed BChE, however, tends to be in monomer or dimer oligomeric forms, which are far less stable than the tetramer form of the enzyme. When BChE is transiently expressed in Nicotiana benthamiana, it is produced mainly as monomers and dimers. However, when the protein is expressed through stable transformation, it produces much greater proportions of tetramers. Tetramerization of WT human plasma derived BChE is facilitated by the binding of a proline rich peptide. In this thesis, I investigated if a putative plant-derived analog of the mammalian proline-rich attachment domain caused stably expressed cocaine hydrolase variants of human BChE to undergo tetramerization. I also examined if co-expression of peptides with known proline-rich attachment domains further shifted the monomer-tetramer ratio toward the tetramer.
ContributorsKendle, Robert Player (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Larrimore, Kathy (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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Description
The development of plant-derived antigens is very promising in vaccine research and the ability to synthesize vaccines cheaply and safely in plant, which can then be ingested, has enormous potential benefits. The goal of this project is to summarize and synthesize the work of current scientists on this issue into

The development of plant-derived antigens is very promising in vaccine research and the ability to synthesize vaccines cheaply and safely in plant, which can then be ingested, has enormous potential benefits. The goal of this project is to summarize and synthesize the work of current scientists on this issue into a cohesive argument in favor of plant-derived vaccinations, while acknowledging any possible drawbacks to their development and the actions that are being taken to overcome them. Hepatitis B, a virus for which orally administered, plant-based vaccines are currently being developed, serves as the case study in which these issues are analyzed. It was found that the synthesized protein is effective immunogenic in humans, but there is still the remaining challenge of making it generate a strong enough immune response through simple ingestion. For this reason, it is clear that plant-derived, oral vaccinations merit further research and hold real prospects of success.
ContributorsCurry, Shannon (Author) / Mason, Hugh (Thesis director) / Escalante, Ananias (Committee member) / LePore, Kate (Committee member) / Barrett, The Honors College (Contributor)
Created2008-05
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Description
Cancer poses a significant burden on the global health system and represents a leading cause of death worldwide. For late-stage cancers, the traditional treatments of chemotherapy, radiation, and surgery are not always viable, and they can pose unnecessary health risks to the patients. New immunotherapies, such as adoptive cell transfer,

Cancer poses a significant burden on the global health system and represents a leading cause of death worldwide. For late-stage cancers, the traditional treatments of chemotherapy, radiation, and surgery are not always viable, and they can pose unnecessary health risks to the patients. New immunotherapies, such as adoptive cell transfer, are being developed and refined to treat such cancers. T cell immunotherapies in particular, where a patient’s T cell lymphocytes are isolated and amplified to be re-infused into the patient or where human cell lines are engineered to express T cell receptors for the recognition of common cancer antigens, are being expanded on because for some cancers, they could be the only option. Constructing an optimal pipeline for cloning and expression of antigen-specific TCRs has significant bearing on the efficacy of engineered cell lines for ACT. Adoptive T cell transfer, while making great strides, has to overcome a diverse T cell repertoire – cloning and expressing antigen-specific TCRs can mediate this understanding. Having identified the high frequency FluM1-specific TCR sequences in stimulated donor PBMCs, it was hypothesized that the antigen-specific TCR could be reconstructed via Gateway cloning methods and tested for expression and functionality. Establishing this pipeline would confirm an ability to properly pair and express the heterodimeric chains. In the context of downstream applications, neoantigens would be used to stimulate T cells, the α and β chains would be paired via single-cell or bulk methods, and instead of Gateway cloning, the CDR3 hypervariable regions α and β chains alone would be co-expressed using Golden Gate assembly methods.
ContributorsHirneise, Gabrielle Rachel (Author) / Anderson, Karen (Thesis director) / Mason, Hugh (Committee member) / Hariadi, Hugh (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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Description
A chimeric, humanized monoclonal antibody that recognizes a highly conserved fusion loop found on flaviviruses was constructed with a geminiviral replicon and transiently expressed in Nicotiana benthamiana plants through Agrobacterium tumefaciens infiltration. Characterization and expression studies were then conducted to confirm correct assembly of the antibody. Once the antibody was

A chimeric, humanized monoclonal antibody that recognizes a highly conserved fusion loop found on flaviviruses was constructed with a geminiviral replicon and transiently expressed in Nicotiana benthamiana plants through Agrobacterium tumefaciens infiltration. Characterization and expression studies were then conducted to confirm correct assembly of the antibody. Once the antibody was purified, an ELISA was conducted to validate that the antibody was able to bind to the flavivirus fusion loop.
ContributorsPardhe, Mary (Author) / Mason, Hugh (Thesis director) / Chen, Qiang (Committee member) / Mor, Tsafrir (Committee member) / School of Life Sciences (Contributor) / Department of Information Systems (Contributor) / W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05