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- Member of: Theses and Dissertations
Description
CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self
onself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy.
onself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy.
ContributorsKrishna, Sri (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Committee member) / Jacobs, Bertram L (Committee member) / Lake, Douglas F (Committee member) / Arizona State University (Publisher)
Created2017
Description
Introduction: Human papillomavirus (HPV) infection is seen in up to 90% of cases of cervical cancer, the third leading cancer cause of death in women. Current HPV screening focuses on only two HPV types and covers roughly 75% of HPV-associated cervical cancers. A protein based assay to test for antibody biomarkers against 98 HPV antigens from both high and low risk types could provide an inexpensive and reliable method to screen for patients at risk of developing invasive cervical cancer. Methods: 98 codon optimized, commercially produced HPV genes were cloned into the pANT7_cGST vector, amplified in a bacterial host, and purified for mammalian expression using in vitro transcription/translation (IVTT) in a luminescence-based RAPID ELISA (RELISA) assay. Monoclonal antibodies were used to determine immune cross-reactivity between phylogenetically similar antigens. Lastly, several protein characteristics were examined to determine if they correlated with protein expression. Results: All genes were successfully moved into the destination vector and 86 of the 98 genes (88%) expressed protein at an adequate level. A difference was noted in expression by gene across HPV types but no correlation was found between protein size, pI, or aliphatic index and expression. Discussion: Further testing is needed to express the remaining 12 HPV genes. Once all genes have been successfully expressed and purified at high concentrations, DNA will be printed on microscope slides to create a protein microarray. This microarray will be used to screen HPV-positive patient sera for antibody biomarkers that may be indicative of cervical cancer and precancerous cervical neoplasias.
ContributorsMeshay, Ian Matthew (Author) / Anderson, Karen (Thesis director) / Magee, Mitch (Committee member) / Katchman, Benjamin (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
In the United States, a dispute has arisen over the safety and need for vaccination, particularly in regard to compulsory vaccination laws. New outlets and social media sites publish countless reports about the dangers of vaccines or of known adverse reactions as well as imagined or unproven worries. Individuals' rights to choose to get vaccinated or allow their children to be vaccinated comes to direct conflict with measures needed to protect communities from preventable viral diseases. The controversy surrounding vaccines is not new, nor necessarily are the fundamental reasons for skepticism. Looking back through the history of vaccines as a medical tool, the evolution of the controversy can be observed taking place with each new historical context, scientific development, and social conditions. Despite scientific research and assurances of vaccine safety, opposition and unease about vaccination appear to take Looking individually at the development and distribution of the smallpox (variola virus), polio (poliovirus) and human papilloma virus(HPV) vaccines, concerns regarding the violation of personal rights, safety of vaccines themselves, and social stigmas and connotations surrounding vaccines can be seen to evolve and change. Due to the way doubt can manifest in different ways over time, it may be impossible to fully end the vaccine debate. However, nderstanding the sociological factors behind anti-vaccine sentiment may allow healthcare professionals to work with concerned people with a particular care to address these visceral and sometimes irrational fears surrounding vaccination.
ContributorsStevens, Luke Christian (Author) / Jacobs, Bertram (Thesis director) / Washo-Krupps, Delon (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Disturbances in the protein interactome often play a large role in cancer progression. Investigation of protein-protein interactions (PPI) can increase our understanding of cancer pathways and will disclose unknown targets involved in cancer disease biology. Although numerous methods are available to study protein interactions, most platforms suffer from drawbacks including high false positive rates, low throughput, and lack of quantification. Moreover, most methods are not compatible for use in a clinical setting. To address these limitations, we have developed a multiplexed, in-solution protein microarray (MISPA) platform with broad applications in proteomics. MISPA can be used to quantitatively profile PPIs and as a robust technology for early detection of cancers. This method utilizes unique DNA barcoding of individual proteins coupled with next generation sequencing to quantitatively assess interactions via barcode enrichment. We have tested the feasibility of this technology in the detection of patient immune responses to oropharyngeal carcinomas and in the discovery of novel PPIs in the B-cell receptor (BCR) pathway. To achieve this goal, 96 human papillomavirus (HPV) antigen genes were cloned into pJFT7-cHalo (99% success) and pJFT7-n3xFlag-Halo (100% success) expression vectors. These libraries were expressed via a cell-free in vitro transcription-translation system with 93% and 96% success, respectively. A small-scale study of patient serum interactions with barcoded HPV16 antigens was performed and a HPV proteome-wide study will follow using additional patient samples. In addition, 15 query proteins were cloned into pJFT7_nGST expression vectors, expressed, and purified with 93% success to probe a library of 100 BCR pathway proteins and detect novel PPIs.
ContributorsRinaldi, Capria Lakshmi (Author) / LaBaer, Joshua (Thesis director) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
This paper describes a thesis project in which I adapted validated survey questions to assess male college students’ perceptions of HPV and the HPV vaccine. Initial studies about HPV were focused on women in order to understand cervical cancer, and it was later discovered that HPV can cause other cancers, even types that can affect men. Because the original research was focused on women, this lead to a delay in research about HPV in males. After the creation of the HPV vaccine, HPV vaccine promotion efforts were historically focused on women, overlooking men as potential targets for this cancer prevention vaccine. This paper briefly describes the HPV vaccine, historic marketing efforts which have focused on vaccinating women against HPV to prevent cervical cancer, and the implications that this presents for men, particularly male college students who are at an increased risk for contracting HPV. College men are an important population to study because of these potential risk factors in addition to discrepancies in their vaccination rates prior to attending a university. The Theory of Planned Behavior (TPB) and Health Belief Model (HBM) have been shown to predict HPV vaccine uptake among college students, including in studies that focus on college males. For this thesis, I identified questions driven by the two health behavior theories, selected and modified questions for survey research, and am currently conducting the survey using REDCap software at Arizona State University. This project serves to further the conversation about men’s health in regards to HPV and the HPV vaccine in addition to addressing the problematic lack of attention on college males within this scope of research.
ContributorsStewart, Vivian Persons (Author) / Koskan, Alexis (Thesis director) / Green, Ellen (Committee member) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Significance Background: Human papilloma virus (HPV) is the most common sexually transmitted infection, affecting 79 million Americans today and an additional 14 million Americans becoming infected with HPV each year. HPV infection may lead to the development of genital warts and several types of cancers including both cervical and oropharyngeal cancers. The promotion of currently available HPV vaccines is important to prevent HPV transmission and reduce the prevalence of the comorbidities associated with infection. Promotion to Vietnamese-Americans in particular is important because of the increased rates of cervical cancers seen in this population. As Vietnamese-American mothers often act as the primary healthcare decision maker for their children, they were chosen as the target population for this intervention. Purpose: This study aims to (1) develop personal digital stories about HPV and HPV vaccination among Vietnamese women with adolescent children who are vaccinated against HPV; and (2) share these stories with a group of Vietnamese American mothers and assess the effect of the stories in changing the attitudes, beliefs, and intention to vaccinate for HPV. Methods: This study used a two-step process to design, implement, and evaluate digital stories to improve Vietnamese mothers' attitudes, beliefs, and intention to vaccinate their adolescent children against HPV. The first step was a formative research design to develop the digital stories. The second step was quasi-experimental with a pre and posttest design to evaluate the effect of the stories. Results: The first phase has produced two digital stories which will be screened recruitment has been completed for phase two. Content analysis showed the importance of community resources, the desire to protect children, a history of familial and/or personal cancer, concerns about side effects, and the influence of healthcare providers as themes in both stories. Recruitment efforts are underway to recruit eligible Vietnamese mothers to assess the effect of these stories. Data collection is ongoing. Conclusions and lessons learned: The project has yielded two digital stories and recruitment for phase two is underway. This project has been successful in obtaining IRB approval, recruiting phase one participants, holding a digital storytelling workshop, designing the phase two survey, and beginning data collection efforts. The phase two recruitment has been challenging and will necessitate a change in strategy to find participants.
ContributorsMartin, Kieran John Patrick (Author) / Chen, Angela Chia-Chen (Thesis director) / Larkey, Linda (Committee member) / Arizona State University. College of Nursing & Healthcare Innovation (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Oropharyngeal cancer (OPC) is the world's sixth most common cancer and in many cases is associated with infection with human papillomavirus (HPV) type 16. Antibodies (Abs) to HPV16 viral antigens are potential diagnostic biomarkers of HPV-associated OPC (HPV OPC). A custom multiplexed bead array assay was used to detect Abs to HPV16 antigens E1, CE2, NE2, E4, E5, E6, E7, L1, and L2. Following extensive optimization of the assay, these genes were expressed as GST-fusion proteins and captured onto anti-GST magnetic beads. Serum was obtained from 256 OPC patients at the time of diagnosis and from 78 healthy controls. The median fluorescent intensity (MFI) was determined for each antigen and ratios of MFI to control GST-fusion protein were determined for each serum sample. Cutoff values were set as the mean + 3 SD of the MFIs of healthy controls and p-values were calculated using Wilcoxon unpaired and Fisher's exact test. Results of this experiment showed that HPV16 E1, CE2, NE2, E4, E6, and E7 Ab levels were elevated in OPC patients compared to controls (p<0.001), as were Ab levels to L1 (p = 0.013) and L2 (p = 0.023), per Fischer's exact test. Abs to CE2, NE2, E6, and E7 were identified as a potential biomarker panel for early detection of HPV OPC. For the 111 patients with known HPV+ tumors as measured by tumor PCR of E6 and/or E7, this assay had a sensitivity of 90% and specificity of 87% (AUC = 0.96). From these results, we conclude that custom bead array assays can be used to detect HPV16 Abs in patient sera, and we have identified a 4-Ab biomarker panel for the early detection of HPV OPC.
ContributorsGoulder, Alison Leigh (Author) / Anderson, Karen (Thesis director) / Lake, Douglas (Committee member) / Cheng, Julia (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
Background: High risk types of human papillomavirus (HPV) are known to cause cancer, including cervical (99%) and oropharyngeal cancer (70%). HPV type 16 is the most common subtype. Three antigens that are critical for integration or tumor progression are E2, E6 and E7. In this study, we developed a systematic approach to identify naturally-processed HPV16-derived HLA class I epitopes for immunotherapy development. Methods: K562 cells, which lack HLA expression, were transduced with each HPV16 antigen using lentivirus and supertransfected with HLA-A2 by nucleofection. Stable cell lines expressing each antigen were selected for and maintained throughout the investigation. In order to establish a Gateway-compatible vector for robust transient gene expression, a Gateway recombination expression cloning cassette was inserted into the commercial Lonza pMAX GFP backbone, which has been experimentally shown to display high transfection expression efficiency. GFP was cloned into the vector and plain K562 cells were transfected with the plasmid by nucleofection. Results: Expression of K562-A2 was tested at various time points by flow cytometry and A2 expression was confirmed. Protein expression was shown for the transduced K562 E7 by Western blot analysis. High transfection efficiency of the pMAX_GFP_Dest vector (up to 97% GFP+ cells) was obtained 48 hours post transfection, comparable to the commercial GFP-plasmid. Conclusion: We have established a rapid system for target viral antigen co-expression with single HLA molecules for analysis of antigen presentation. Using HPV as a model system, our goal is to identify specific antigenic peptide sequences to develop immunotherapeutic treatments for HPV-associated cancers.
ContributorsVarda, Bianca Marie (Author) / Anderson, Karen (Thesis director) / Borges, Chad (Committee member) / Krishna, Sri (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Portable health diagnostic systems seek to perform medical grade diagnostics in non-ideal environments. This work details a robust fault tolerant portable health diagnostic design implemented in hardware, firmware and software for the detectionof HPV in low-income countries. The device under device under test (DUT) is a fluorescence based lateral flow assay (LFA) point-of-care (POC) device. This work’s contributions are: firmware and software development, calibration routine implementation, device performance characterization and a proposed method of in-software fault detection. Firmware was refactored from the original implementation of the POC fluorescence reader to expose an application programming interface (API) via USB. Companion software available for desktop environments (Windows, Mac and Linux) was created to interface with this firmware API and conduct macro level routines to request and receive fluorescence data while presenting a user-friendly interface to clinical technicians. Lastly, an environmental chamber was constructed to conduct sequential diagnostic reads in order to observe sensor drift and other deviations that might present themselves in real-world usage. The results from these evaluations show a standard deviation of less than 1% in fluorescence readings in nominal temperature environments (approx. 25C) suggesting that this system will have a favorable signal-to-noise (SNR) ratio in such a setting. In non-ideal over heated environments (≥38C), the evaluation results showed performance degradation with standard deviations as large as 15%.
ContributorsLue Sang, Christopher David (Author) / Blain Christen, Jennifer M (Thesis advisor) / Ozev, Sule (Committee member) / Goryll, Michael (Committee member) / Raupp, Gregory (Committee member) / Arizona State University (Publisher)
Created2022
Description
Native American COVID vaccination trends have deviated from the vaccinated trends for other vaccines such as the HPV and flu. Native Americans are reporting the highest vaccinations in the nation compared to all racial and ethnic groups in the country. Despite this, some are still hesitant in taking the vaccine. This study investigates the factors that influence vaccine uptake and hesitancy among Native American Youth in Arizona. The study utilizes data from the Arizona Youth Identity Project, a mixed-methods study examining young adults’ political engagement and sense of identity as Americans. 10 out of 13 individuals at the time of the study had received the COVID vaccine. Three themes, trust, knowledge, and social responsibility were identified. Most of the reasons for hesitancy came from distrust of government and biomedicine. Most participants had no knowledge of vaccine effectiveness, mechanism or effects but viewed getting vaccinated as a social responsibility to protect their families and communities. These findings indicate that the main goal of vaccinations among Native American youth was to protect the most vulnerable elders.
ContributorsAbaidoo, Joy (Author) / Gonzales, Angela (Thesis director) / Doebbeling, Bradley (Committee member) / Barrett, The Honors College (Contributor) / Hugh Downs School of Human Communication (Contributor) / School of Life Sciences (Contributor)
Created2022-05