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- All Subjects: Immunology
- Creators: LaBaer, Joshua
Vaccines are modern medicine’s best way of combating the majority of viral and bacterial illnesses and contagions to date. Thanks to the introduction of vaccines since the first uses of them in 1796 (Jenner’s smallpox vaccine), they have drastically reduced figures of disease worldwide, turning once lethal and life changing conditions into minor annoyances; Some of these afflictions have even become nonexistent or even extinct in certain parts of the world outside of a controlled laboratory setting. With many advancements and overwhelming evidence proving their efficiency, it is clear that vaccines have become nothing less than a necessity for everyday healthcare in today’s world. <br/>The greatest contributor to the creation and evolution of vaccines throughout the years is by far the progress and work done in the field of molecular and cellular biology. These advancements have become the bedrock of modern vaccination, as shown by the differing types of vaccines and their methodology. The most common varieties of vaccines are include ‘dead’ or inactivated vaccines, one such example being the pertussis strain of vaccines, which have either dead or torn apart cells for the body to easily fight off, allowing the immune system to easily and quickly counter the illness; Additionally, there are also live attenuated vaccines (LAVs) in which a weaker version of the pathogen is introduced to the body to stimulate an immune response, or a recombinant mRNA vaccine where mRNA containing the coding for an antigen is presented for immunological response, the latter being what the current COVID-19 vaccines are based on. This is in part aided by the presence of immunological adjuvants, antigens and substances that the immune system can recognize, target, and remember for future infections. However, for more serious illnesses the body needs a bigger threat to analyze, which leads to live vaccines- instead of dead or individual components of a potential pathogen, a weakened version is created in the lab to allow the body to combat it. The idea behind this is the same, but to a larger degree so a more serious illness such as measles, mumps, and rubella (MMR) do not infect us.<br/>However, for the past couple of decades the public’s views on vaccination has greatly varied, with the rise of fear and disinformation leading those to believe that modern medicine is a threat in disguise. The largest of these arguments began in the late 90’s, when Dr. Andrew Wakefield published an article under the Lancet with false information connecting vaccinations to the occurrence of autism in younger children- a theory which has since then been proven incorrect numerous times over. Unfortunately, the rise of hysteria and paranoia in people, along with more misinformation from misleading sources, have strengthened the anti-vaccination cause and has made it into a serious threat to the health of those world-wide.<br/>The aim of this thesis is to provide an accurate and thorough analysis on these three themes- the history of vaccines, their inner workings and machinations in providing immune defenses for the body, and the current controversy of the anti-vaccination movement. Additionally, there will be two other sections going in-depth on two specific areas where vaccination is highly important; The spread and fear of the Human Immunodeficiency Virus (HIV) has been around for nearly four decades, so it begs the question: what makes this such a difficult virus, and how can a vaccine be created to combat it? Additionally, in the last year the world has encountered a new virus that has evolved into a global pandemic, SARS-COV 2. This new strain of coronavirus has shown itself to be highly contagious and rapidly mutating, and the race to quickly develop a vaccine to counteract it has been on-going since its first major infections in Wuhan, China. Overall, this thesis will go in-depth in providing the most accurate, up-to-date, and critical information regarding vaccinations today.
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.
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.
To characterized these subtypes, an in vitro cytokine induced type 1 (E1) and type 2 (E2) eosinophil model was developed that display features and functions of eosinophils found in vivo. For example, E1 eosinophils secrete type 1 mediators (e.g., IL-12, CXCL9 and CXCL10), express iNOS and express increased levels of the surface molecules PDL1 and MHC-I. Conversely, E2 eosinophils release type 2 mediators (e.g., IL4, IL13, CCL17, and CCL22), degranulate and express increased surface molecules CD11b, ST2 and Siglec-F. Completion of differential expression analysis of RNAseq on these subtypes revealed 500 and 655 unique genes were upregulated in E1 and E2 eosinophils, respectively. Functional enrichment studies showed interferon regulatory factor (IRF) transcription factors were uniquely regulated in both mouse and human E1 and E2 eosinophils. These subtypes are sensitive to their environment, modulating their IRF and cell surface expression when stimulated with opposing cytokines, suggesting plasticity.
To identify and study these subtypes in situ, chromogenic and fluorescent eosinophil-specific immunostaining protocols were developed. Methods were created and optimized, here, to identify eosinophils by their granule proteins in formalin fixed mouse tissues. Yet, eosinophil-specific antibodies alone are not enough to identify and study the complex interactions eosinophil subtypes perform within a tissue. Therefore, as part of this thesis, a novel highly-multiplexed immunohistochemistry technique was developed utilizing cleavable linkers to address these concerns. This technique is capable of analyzing up to 22 markers within a single biopsy with single-cell resolution. With this approach, eosinophil subtypes can be studied in situ in routine patient biopsies.