Matching Items (15)
Filtering by
- All Subjects: Immunology
- All Subjects: Plant Biology
- Creators: Mason, Hugh
- Creators: Chen, Qiang
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
Immunotherapy has been revitalized with the advent of immune checkpoint blockade
treatments, and neo-antigens are the targets of immune system in cancer patients who
respond to the treatments. The cancer vaccine field is focused on using neo-antigens from
unique point mutations of genomic sequence in the cancer patient for making
personalized cancer vaccines. However, we choose a different path to find frameshift
neo-antigens at the mRNA level and develop broadly effective cancer vaccines based on
frameshift antigens.
In this dissertation, I have summarized and characterized all the potential frameshift
antigens from microsatellite regions in human, dog and mouse. A list of frameshift
antigens was validated by PCR in tumor samples and the mutation rate was calculated for
one candidate – SEC62. I develop a method to screen the antibody response against
frameshift antigens in human and dog cancer patients by using frameshift peptide arrays.
Frameshift antigens selected by positive antibody response in cancer patients or by MHC
predictions show protection in different mouse tumor models. A dog version of the
cancer vaccine based on frameshift antigens was developed and tested in a small safety
trial. The results demonstrate that the vaccine is safe and it can induce strong B and T cell
immune responses. Further, I built the human exon junction frameshift database which
includes all possible frameshift antigens from mis-splicing events in exon junctions, and I
develop a method to find potential frameshift antigens from large cancer
immunosignature dataset with these databases. In addition, I test the idea of ‘early cancer
diagnosis, early treatment’ in a transgenic mouse cancer model. The results show that
ii
early treatment gives significantly better protection than late treatment and the correct
time point for treatment is crucial to give the best clinical benefit. A model for early
treatment is developed with these results.
Frameshift neo-antigens from microsatellite regions and mis-splicing events are
abundant at mRNA level and they are better antigens than neo-antigens from point
mutations in the genomic sequences of cancer patients in terms of high immunogenicity,
low probability to cause autoimmune diseases and low cost to develop a broadly effective
vaccine. This dissertation demonstrates the feasibility of using frameshift antigens for
cancer vaccine development.
treatments, and neo-antigens are the targets of immune system in cancer patients who
respond to the treatments. The cancer vaccine field is focused on using neo-antigens from
unique point mutations of genomic sequence in the cancer patient for making
personalized cancer vaccines. However, we choose a different path to find frameshift
neo-antigens at the mRNA level and develop broadly effective cancer vaccines based on
frameshift antigens.
In this dissertation, I have summarized and characterized all the potential frameshift
antigens from microsatellite regions in human, dog and mouse. A list of frameshift
antigens was validated by PCR in tumor samples and the mutation rate was calculated for
one candidate – SEC62. I develop a method to screen the antibody response against
frameshift antigens in human and dog cancer patients by using frameshift peptide arrays.
Frameshift antigens selected by positive antibody response in cancer patients or by MHC
predictions show protection in different mouse tumor models. A dog version of the
cancer vaccine based on frameshift antigens was developed and tested in a small safety
trial. The results demonstrate that the vaccine is safe and it can induce strong B and T cell
immune responses. Further, I built the human exon junction frameshift database which
includes all possible frameshift antigens from mis-splicing events in exon junctions, and I
develop a method to find potential frameshift antigens from large cancer
immunosignature dataset with these databases. In addition, I test the idea of ‘early cancer
diagnosis, early treatment’ in a transgenic mouse cancer model. The results show that
ii
early treatment gives significantly better protection than late treatment and the correct
time point for treatment is crucial to give the best clinical benefit. A model for early
treatment is developed with these results.
Frameshift neo-antigens from microsatellite regions and mis-splicing events are
abundant at mRNA level and they are better antigens than neo-antigens from point
mutations in the genomic sequences of cancer patients in terms of high immunogenicity,
low probability to cause autoimmune diseases and low cost to develop a broadly effective
vaccine. This dissertation demonstrates the feasibility of using frameshift antigens for
cancer vaccine development.
ContributorsZhang, Jian (Author) / Johnston, Stephen Albert (Thesis advisor) / Chang, Yung (Committee member) / Stafford, Phillip (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2018
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
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
Description
Is it possible to treat the mouth as a natural environment, and determine new methods to keep the microbiome in check? The need for biodiversity in health may suggest that every species carries out a specific function that is required to maintain equilibrium and homeostasis within the oral cavity. Furthermore, the relationship between the microbiome and its host is mutually beneficial because the host is providing microbes with an environment in which they can flourish and, in turn, keep their host healthy. Reviewing examples of larger scale environmental shifts could provide a window by which scientists can make hypotheses. Certain medications and healthcare treatments have been proven to cause xerostomia. This disorder is characterized by a dry mouth, and known to be associated with a change in the composition, and reduction, of saliva. Two case studies performed by Bardow et al, and Leal et al, tested and studied the relationships of certain medications and confirmed their side effects on the salivary glands [2,3]. Their results confirmed a relationship between specific medicines, and the correlating complaints of xerostomia. In addition, Vissink et al conducted case studies that helped to further identify how radiotherapy causes hyposalivation of the salivary glands [4]. Specifically patients that have been diagnosed with oral cancer, and are treated by radiotherapy, have been diagnosed with xerostomia. As stated prior, studies have shown that patients having an ecologically balanced and diverse microbiome tend to have healthier mouths. The oral cavity is like any biome, consisting of commensalism within itself and mutualism with its host. Due to the decreased salivary output, caused by xerostomia, increased parasitic bacteria build up within the oral cavity thus causing dental disease. Every human body contains a personalized microbiome that is essential to maintaining health but capable of eliciting disease. The Human Oral Microbiomics Database (HOMD) is a set of reference 16S rRNA gene sequences. These are then used to define individual human oral taxa. By conducting metagenomic experiments at the molecular and cellular level, scientists can identify and label micro species that inhabit the mouth during parasitic outbreaks or a shifting of the microbiome. Because the HOMD is incomplete, so is our ability to cure, or prevent, oral disease. The purpose of the thesis is to research what is known about xerostomia and its effects on the complex microbiome of the oral cavity. It is important that researchers determine whether this particular perspective is worth considering. In addition, the goal is to create novel experiments for treatment and prevention of dental diseases.
ContributorsHalcomb, Michael Jordan (Author) / Chen, Qiang (Thesis director) / Steele, Kelly (Committee member) / Barrett, The Honors College (Contributor) / College of Letters and Sciences (Contributor)
Created2015-05
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, 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
Description
Influenza is a deadly disease for which effective vaccines are sorely lacking. This is largely due to the phenomena of antigenic shift and drift in the influenza virus's surface proteins, hemagglutinin (HA) and neuraminidase (NA). The ectodomain of the matrix 2 protein (M2e) of influenza A, however, has demonstrated high levels of conservation. On its own it is poorly immunogenic and offers little protection against influenza infections, but by combining it with a potent adjuvant, this limitation may be overcome. Recombinant immune complexes, or antigens fused to antibodies that have been engineered to form incredibly immunogenic complexes with one another, were previously shown to be useful, immunogenic platforms for the presentation of various antigens and could provide the boost in immunogenicity that M2e needs to become a powerful universal influenza A vaccine. In this thesis, genetic constructs containing geminiviral replication proteins and coding for a consensus sequence of dimeric M2e fused to antibodies featuring complimentary epitopes and epitope tags were generated and used to transform Agrobacterium tumefaciens. The transformed bacteria was then used to cause Nicotiana benthamiana to transiently express M2e-RICs at very high levels, with enough RICs being gathered to evaluate their potency in future mouse trials. Future directions and areas for further research are discussed.
ContributorsFavre, Brandon Chetan (Author) / Mason, Hugh (Thesis director) / Mor, Tsafrir (Committee member) / Diamos, Andrew (Committee member) / Department of Psychology (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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
The expression of complex proteins was studied in multiple plant systems. Recombinant spider silk, which could be utilized for biomedical applications such as coatings or doped into silk fibers, was successfully expressed in Nicotiana. benthamiana wild type and GnGn glycoengineered transgenic plants and purified from endogenous plant proteins which could be utilized for biomedical applications such as coatings or doped into silk fibers. However, the purification process requires further optimization to result in commercialized production of recombinant spider silk. Green fluorescent protein and Norovirus virus-like particles were expressed in multiple plant systems including alfalfa, beets, lettuce, and spinach, in addition to N. benthamiana, to determine the ability of these plant expression systems to produce vaccine candidates for edible vaccine applications in the agricultural sector as well as low-to-middle income countries. It was determined that alfalfa, beets, and lettuce are potential high production expression systems for edible vaccines however they require further optimization to be commercialized. Lastly, novel virus-like particles and antigen presenting nanoparticles based on the bacteriophage AP205 coat protein and norovirus capsid proteins fused to human papillomavirus L2 protein segments (S and P) were expressed in N. benthamiana and utilized to vaccinate mice against the L2 capsid protein (aa14-38x2 and aa14-122) of Human Papillomavirus 16 to study a potential boosting effect of the Recombinant Immune Complex vaccine platform upon prime-boost dosing with the virus-like particle being the prime and the Recombinant Immune Complex being the boost in this vaccine schema.
ContributorsHunter, Joseph G (Author) / Mason, Hugh (Thesis advisor) / Arizona State University (Publisher)
Created2023