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- All Subjects: Immunology
- Creators: Chang, Yung
- Creators: Stafford, Phillip
Description
Cancer is one of the most serious global diseases. We have focused on cancer immunoprevention. My thesis projects include developing a prophylactic primary and metastatic cancer vaccines, early cancer detection and investigation of genes involved in tumor development. These studies were focused on frame-shift (FS) antigens. The FS antigens are generated by genomic mutations or abnormal RNA processing, which cause a portion of a normal protein to be translated out of frame. The concept of the prophylactic cancer vaccine is to develop a general cancer vaccine that could prevent healthy people from developing different types of cancer. We have discovered a set of cancer specific FS antigens. One of the FS candidates, structural maintenance of chromosomes protein 1A (SMC1A) FS, could start to accumulate at early stages of tumor and be specifically exposed to the immune system by tumor cells. Prophylactic immunization with SMC1A-FS could significantly inhibit primary tumor development in different murine tumor models and also has the potential to inhibit tumor metastasis. The SMC1A-FS transcript was detected in the plasma of the 4T1/BALB/c mouse tumor model. The tumor size was correlated with the transcript ratio of the SMC1A-FS verses the WT in plasma, which could be measured by regular RT-PCR. This unique cancer biomarker has a practical potential for a large population cancer screen, as well as clinical tumor monitoring. With a set of mimotope peptides, antibodies against SMC1A-FS peptide were detected in different cancer patients, including breast cancer, pancreas cancer and lung cancer with a 53.8%, 56.5% and 12.5% positive rate respectively. This suggested that the FS antibody could be a biomarker for early cancer detection. The characterization of SMC1A suggested that: First, the deficiency of the SMC1A is common in different tumors and able to promote tumor initiation and development; second, the FS truncated protein may have nucleolus function in normal cells. Mis-control of this protein may promote tumor development. In summary, we developed a systematic general cancer prevention strategy through the variety immunological and molecular methods. The results gathered suggest the SMC1A-FS may be useful for the detection and prevention of cancer.
ContributorsShen, Luhui (Author) / Johnston, Stephen Albert (Thesis advisor) / Chang, Yung (Committee member) / Miller, Laurence (Committee member) / Sykes, Kathryn (Committee member) / Jacobs, Bertram (Committee member) / Arizona State University (Publisher)
Created2012
Description
The advent of new high throughput technology allows for increasingly detailed characterization of the immune system in healthy, disease, and age states. The immune system is composed of two main branches: the innate and adaptive immune system, though the border between these two states is appearing less distinct. The adaptive immune system is further split into two main categories: humoral and cellular immunity. The humoral immune response produces antibodies against specific targets, and these antibodies can be used to learn about disease and normal states. In this document, I use antibodies to characterize the immune system in two ways: 1. I determine the Antibody Status (AbStat) from the data collected from applying sera to an array of non-natural sequence peptides, and demonstrate that this AbStat measure can distinguish between disease, normal, and aged samples as well as produce a single AbStat number for each sample; 2. I search for antigens for use in a cancer vaccine, and this search results in several candidates as well as a new hypothesis. Antibodies provide us with a powerful tool for characterizing the immune system, and this natural tool combined with emerging technologies allows us to learn more about healthy and disease states.
ContributorsWhittemore, Kurt (Author) / Sykes, Kathryn (Thesis advisor) / Johnston, Stephen A. (Committee member) / Jacobs, Bertram (Committee member) / Stafford, Phillip (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2014
Description
Peptide microarrays are to proteomics as sequencing is to genomics. As microarrays become more content-rich, higher resolution proteomic studies will parallel deep sequencing of nucleic acids. Antigen-antibody interactions can be studied at a much higher resolution using microarrays than was possible only a decade ago. My dissertation focuses on testing the feasibility of using either the Immunosignature platform, based on non-natural peptide sequences, or a pathogen peptide microarray, which uses bioinformatically-selected peptides from pathogens for creating sensitive diagnostics. Both diagnostic applications use relatively little serum from infected individuals, but each approaches diagnosis of disease differently. The first project compares pathogen epitope peptide (life-space) and non-natural (random-space) peptide microarrays while using them for the early detection of Coccidioidomycosis (Valley Fever). The second project uses NIAID category A, B and C priority pathogen epitope peptides in a multiplexed microarray platform to assess the feasibility of using epitope peptides to simultaneously diagnose multiple exposures using a single assay. Cross-reactivity is a consistent feature of several antigen-antibody based immunodiagnostics. This work utilizes microarray optimization and bioinformatic approaches to distill the underlying disease specific antibody signature pattern. Circumventing inherent cross-reactivity observed in antibody binding to peptides was crucial to achieve the goal of this work to accurately distinguishing multiple exposures simultaneously.
ContributorsNavalkar, Krupa Arun (Author) / Johnston, Stephen A. (Thesis advisor) / Stafford, Phillip (Thesis advisor) / Sykes, Kathryn (Committee member) / Jacobs, Bertram (Committee member) / Arizona State University (Publisher)
Created2014
Description
Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by progressive autoimmune destruction of insulin-producing pancreatic β-cells. Genetic, immunological and environmental factors contribute to T1D development. The focus of this dissertation is to track the humoral immune response in T1D by profiling autoantibodies (AAbs) and anti-viral antibodies using an innovative protein array platform called Nucleic Acid Programmable Protein Array (NAPPA).
AAbs provide value in identifying individuals at risk, stratifying patients with different clinical courses, improving our understanding of autoimmune destructions, identifying antigens for cellular immune response and providing candidates for prevention trials in T1D. A two-stage serological AAb screening against 6,000 human proteins was performed. A dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) was validated with 36% sensitivity at 98% specificity by an orthogonal immunoassay. This is the first systematic screening for novel AAbs against large number of human proteins by protein arrays in T1D. A more comprehensive search for novel AAbs was performed using a knowledge-based approach by ELISA and a screening-based approach against 10,000 human proteins by NAPPA. Six AAbs were identified and validated with sensitivities ranged from 16% to 27% at 95% specificity. These two studies enriched the T1D “autoantigenome” and provided insights into T1D pathophysiology in an unprecedented breadth and width.
The rapid rise of T1D incidence suggests the potential involvement of environmental factors including viral infections. Sero-reactivity to 646 viral antigens was assessed in new-onset T1D patients. Antibody positive rate of EBV was significantly higher in cases than controls that suggested a potential role of EBV in T1D development. A high density-NAPPA platform was demonstrated with high reproducibility and sensitivity in profiling anti-viral antibodies.
This dissertation shows the power of a protein-array based immunoproteomics approach to characterize humoral immunoprofile against human and viral proteomes. The identification of novel T1D-specific AAbs and T1D-associated viruses will help to connect the nodes in T1D etiology and provide better understanding of T1D pathophysiology.
AAbs provide value in identifying individuals at risk, stratifying patients with different clinical courses, improving our understanding of autoimmune destructions, identifying antigens for cellular immune response and providing candidates for prevention trials in T1D. A two-stage serological AAb screening against 6,000 human proteins was performed. A dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) was validated with 36% sensitivity at 98% specificity by an orthogonal immunoassay. This is the first systematic screening for novel AAbs against large number of human proteins by protein arrays in T1D. A more comprehensive search for novel AAbs was performed using a knowledge-based approach by ELISA and a screening-based approach against 10,000 human proteins by NAPPA. Six AAbs were identified and validated with sensitivities ranged from 16% to 27% at 95% specificity. These two studies enriched the T1D “autoantigenome” and provided insights into T1D pathophysiology in an unprecedented breadth and width.
The rapid rise of T1D incidence suggests the potential involvement of environmental factors including viral infections. Sero-reactivity to 646 viral antigens was assessed in new-onset T1D patients. Antibody positive rate of EBV was significantly higher in cases than controls that suggested a potential role of EBV in T1D development. A high density-NAPPA platform was demonstrated with high reproducibility and sensitivity in profiling anti-viral antibodies.
This dissertation shows the power of a protein-array based immunoproteomics approach to characterize humoral immunoprofile against human and viral proteomes. The identification of novel T1D-specific AAbs and T1D-associated viruses will help to connect the nodes in T1D etiology and provide better understanding of T1D pathophysiology.
ContributorsBian, Xiaofang (Author) / LaBaer, Joshua (Thesis advisor) / Mandarino, Lawrence (Committee member) / Chang, Yung (Committee member) / Arizona State University (Publisher)
Created2015
Description
African Swine Fever (ASF), endemic in many African countries, is now spreading to other continents. Though ASF is capable of incurring serious economic losses in affected countries, no vaccine exists to provide immunity to animals. Disease control relies largely on rapid diagnosis and the implementation of movement restrictions and strict eradication programs. Developing a scalable, accurate and low cost diagnostic for ASF will be of great help for the current situation. CIM's 10K random peptide microarray is a new high-throughput platform that allows systematic investigations of immune responses associated with disease and shows promise as a diagnostic tool. In this study, this new technology was applied to characterize the immune responses of ASF virus (ASFV) infections and immunizations. Six sets of sera from ASFV antigen immunized pigs, 6 sera from infected pigs and 20 sera samples from unexposed pigs were tested and analyzed statistically. Results show that both ASFV antigen immunized pigs and ASFV viral infected pigs can be distinguished from unexposed pigs. Since it appears that immune responses to other viral infections are also distinguishable on this platform, it holds the potential of being useful in developing a new ASF diagnostic. The ability of this platform to identify specific ASFV antibody epitopes was also explored. A subtle motif was found to be shared among a set of peptides displaying the highest reactivity for an antigen specific antibody. However, this motif does not seem to match with any antibody epitopes predicted by a linear antibody epitope prediction.
ContributorsXiao, Liang (Author) / Sykes, Kathryn (Thesis advisor) / Zhao, Zhan-Gong (Committee member) / Stafford, Phillip (Committee member) / Arizona State University (Publisher)
Created2011
Description
The Philadelphia chromosome in humans, is on oncogenic translocation between chromosomes 9 and 22 that gives rise to the fusion protein BCR-Abl. This protein is constitutively active resulting in rapid and uncontrolled cell growth in affected cells. The BCR-Abl protein is the hallmark feature of chronic myeloid leukemia (CML) and is seen in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cases. Currently, the first line of treatment is the Abl specific inhibitor Imatinib. Some patients will, however, develop resistance to Imatinib. Research has shown how transformation of progenitor B cells with v-Abl, an oncogene expressed by the Abelson murine leukemia virus, causes rapid proliferation, prevents further differentiation and produces a potentially malignant transformation. We have used progenitor B cells transformed with a temperature-sensitive form of the v-Abl protein that allows us to inactivate or re-activate v-Abl by shifting the incubation temperature. We are trying to use this line as a model to study both the progression from pre-malignancy to malignancy in CML and Imatinib resistance in Ph+ ALL and CML. These progenitor B cells, once v-Abl is reactivated, in most cases, will not return to their natural cell cycle. In this they resemble Ph+ ALL and CML under Imatinib treatment. With some manipulation these cells can break this prolonged G1 arrested phenotype and become a malignant cell line and resistant to Imatinib treatment. Cellular senescence can be a complicated process requiring inter-play between a variety of players. It serves as an alternate option to apoptosis, in that the cell loses proliferative potential, but does not die. Treatment with some cancer therapeutics will induce senescence in some cancers. Such is the case with Imatinib treatment of CML and Ph+ ALL. By using the S9 cell line we have been able to explore the possible routes for breaking of prolonged G1 arrest in these Ph+ leukemias. We inhibited the DNA damage sensor protein ataxia telangiectasia mutated (ATM) and found that prolonged G1 arrest in our S9 cells was broken. While previous research has suggested that the DNA damage sensor protein ataxia-telangiectasia mutated (ATM) has little impact in CML, our research indicates that ATM may play a role in either senescence induction or release.
ContributorsDixon, Sarah E (Author) / Chang, Yung (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Touchman, Jeffrey (Committee member) / Arizona State University (Publisher)
Created2011
Description
The goal of this thesis is to test whether Alzheimer's disease (AD) is associated with distinctive humoral immune changes that can be detected in plasma and tracked across time. This is relevant because AD is the principal cause of dementia, and yet, no specific diagnostic tests are universally employed in clinical practice to predict, diagnose or monitor disease progression. In particular, I describe herein a proteomic platform developed at the Center for Innovations in Medicine (CIM) consisting of a slide with 10.000 random-sequence peptides printed on its surface, which is used as the solid phase of an immunoassay where antibodies of interest are allowed to react and subsequently detected with a labeled secondary antibody. The pattern of antibody binding to the microarray is unique for each individual animal or person. This thesis will evaluate the versatility of the microarray platform and how it can be used to detect and characterize the binding patterns of antibodies relevant to the pathophysiology of AD as well as the plasma samples of animal models of AD and elderly humans with or without dementia. My specific aims were to evaluate the emergence and stability of immunosignature in mice with cerebral amyloidosis, and characterize the immunosignature of humans with AD. Plasma samples from APPswe/PSEN1-dE9 transgenic mice were evaluated longitudinally from 2 to 15 months of age to compare the evolving immunosignature with non-transgenic control mice. Immunological variation across different time-points was assessed, with particular emphasis on time of emergence of a characteristic pattern. In addition, plasma samples from AD patients and age-matched individuals without dementia were assayed on the peptide microarray and binding patterns were compared. It is hoped that these experiments will be the basis for a larger study of the diagnostic merits of the microarray-based immunoassay in dementia clinics.
ContributorsRestrepo Jimenez, Lucas (Author) / Johnston, Stephen A. (Thesis advisor) / Chang, Yung (Committee member) / Reiman, Eric (Committee member) / Sierks, Michael (Committee member) / Arizona State University (Publisher)
Created2011
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
There are many biological questions that require single-cell analysis of gene sequences, including analysis of clonally distributed dimeric immunoreceptors on lymphocytes (T cells and B cells) and/or the accumulation of driver/accessory mutations in polyclonal tumors. Lysis of bulk cell populations results in mixing of gene sequences, making it impossible to know which pairs of gene sequences originated from any particular cell and obfuscating analysis of rare sequences within large populations. Although current single-cell sorting technologies can be used to address some of these questions, such approaches are expensive, require specialized equipment, and lack the necessary high-throughput capacity for comprehensive analysis. Water-in-oil emulsion approaches for single cell sorting have been developed but droplet-based single-cell lysis and analysis have proven inefficient and yield high rates of false pairings. Ideally, molecular approaches for linking gene sequences from individual cells could be coupled with next-generation high-throughput sequencing to overcome these obstacles, but conventional approaches for linking gene sequences, such as by transfection with bridging oligonucleotides, result in activation of cellular nucleases that destroy the template, precluding this strategy. Recent advances in the synthesis and fabrication of modular deoxyribonucleic acid (DNA) origami nanostructures have resulted in new possibilities for addressing many current and long-standing scientific and technical challenges in biology and medicine. One exciting application of DNA nanotechnology is the intracellular capture, barcode linkage, and subsequent sequence analysis of multiple messenger RNA (mRNA) targets from individual cells within heterogeneous cell populations. DNA nanostructures can be transfected into individual cells to capture and protect mRNA for specific expressed genes, and incorporation of origami-specific bowtie-barcodes into the origami nanostructure facilitates pairing and analysis of mRNA from individual cells by high-throughput next-generation sequencing. This approach is highly modular and can be adapted to virtually any two (and possibly more) gene target sequences, and therefore has a wide range of potential applications for analysis of diverse cell populations such as understanding the relationship between different immune cell populations, development of novel immunotherapeutic antibodies, or improving the diagnosis or treatment for a wide variety of cancers.
ContributorsSchoettle, Louis (Author) / Blattman, Joseph N (Thesis advisor) / Yan, Hao (Committee member) / Chang, Yung (Committee member) / Lindsay, Stuart (Committee member) / Arizona State University (Publisher)
Created2017
Description
DNA nanotechnology has been a rapidly growing research field in the recent decades, and there have been extensive efforts to construct various types of highly programmable and robust DNA nanostructures. Due to the advantage that DNA nanostructure can be used to organize biochemical molecules with precisely controlled spatial resolution, herein we used DNA nanostructure as a scaffold for biological applications. Targeted cell-cell interaction was reconstituted through a DNA scaffolded multivalent bispecific aptamer, which may lead to promising potentials in tumor therapeutics. In addition a synthetic vaccine was constructed using DNA nanostructure as a platform to assemble both model antigen and immunoadjuvant together, and strong antibody response was demonstrated in vivo, highlighting the potential of DNA nanostructures to serve as a new platform for vaccine construction, and therefore a DNA scaffolded hapten vaccine is further constructed and tested for its antibody response. Taken together, my research demonstrated the potential of DNA nanostructure to serve as a general platform for immunological applications.
ContributorsLiu, Xiaowei (Author) / Liu, Yan (Thesis advisor) / Chang, Yung (Thesis advisor) / Yan, Hao (Committee member) / Allen, James (Committee member) / Zhang, Peiming (Committee member) / Arizona State University (Publisher)
Created2012