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Vaccinia virus (VACV) is the current vaccine for the highly infectious smallpox disease. Since the eradication of smallpox, VACV has been developed extensively as a heterologous vaccine vector for several pathogens. However, due to the complications associated with this replication competent virus, the safety and efficacy of VACV vaccine vector has been reevaluated. To evaluate the safety and efficacy of VACV, we study the interactions between VACV and the host innate immune system, especially the type I interferon (IFN) signaling pathways. In this work, we evaluated the role of protein kinase R (PKR) and Adenosine Deaminase Acting on RNA 1(ADAR1), which are induced by IFN, in VACV infection. We found that PKR is necessary but is not sufficient to activate interferon regulatory factor 3 (IRF3) in the induction of type I IFN; and the activation of the stress-activated protein kinase/ c-Jun NH2-terminal kinase is required for the PKR-dependent activation of IRF3 during VACV infection. Even though PKR was found to have an antiviral effect in VACV, ADAR1 was found to have a pro-viral effect by destabilizing double stranded RNA (dsRNA), rescuing VACVΔE3L, VACV deleted of the virulence factor E3L, when provided in trans. With the lessons we learned from VACV and host cells interaction, we have developed and evaluated a safe replication-competent VACV vaccine vector for HIV. Our preliminary results indicate that our VACV vaccine vector can still induce the IFN pathway while maintaining the ability to replicate and to express the HIV antigen efficiently. This suggests that this VACV vector can be used as a safe and efficient vaccine vector for HIV.
ContributorsHuynh, Trung Phuoc (Author) / Jacobs, Bertram L (Thesis advisor) / Hogue, Brenda (Committee member) / Chang, Yung (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2013
Description
As advanced as current cancer therapeutics are, there are still challenges that need to be addressed. One of them is the non-specific killing of normal cells in addition to cancerous cells. Ideal cancer therapeutics should be targeted specifically toward tumor cells. Due to the robust self-assembly and versatile addressability of DNA-nanostructures, a DNA tetrahedron nanostructure was explored as a drug carrier. The nanostructure can be decorated with various molecules to either increase immunogenicity, toxicity, or affinity to a specific cell type. The efficiency of the specific binding and internalization of the chosen molecules was measured via flow cytometry. Using a murine B cell lymphoma as the model system, several targeting molecules have been evaluated for their specific binding and induced internalization of DNA nanostructures, including an anti-Igκ antibody, an idiotype-binding peptide, and a g-quadruplex nucleolin specific aptamer. It was found that adding the anti-Igκ antibody appeared to provide increased binding and facilitated cellular internalization. Also, it was found that the presence of CpG appeared to aid in the binding of nanostructures decorated with other molecules, as compared to nanostructures without CpG. The g-quadruplex aptamer thought to specifically bind cancer cells that overexpress nucleolin was tested and found to have better binding to cells when linked to the nanostructure than when alone. The drug doxorubicin was used to load the DNA-nanostructure and attempt to inhibit cancer cell growth. The DNA-nanostructure has the benefit of being self-assembled and customizable, and it has been shown to bind to and internalize into a cancer cell line. The next steps are to test the toxicity of the nanostructure as well as its specificity for cancerous cells compared to noncancerous cells. Furthermore, once those tests are completed the structure’s drug delivery capacity will be tested in tumor bearing mice. The DNA-nanostructure exhibits potential as a cancer specific therapeutic.
ContributorsGomez, Amber Marie (Author) / Chang, Yung (Thesis director) / Anderson, Karen (Committee member) / Liu, Xiaowei (Committee member) / Sanford School of Social and Family Dynamics (Contributor) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Despite the safe and effective use of attenuated vaccines for over fifty years, measles virus (MV) remains an insidious threat to global health. Problematically, infants less than one year of age, who are the most prone to severe infection and death by measles, cannot be immunized using current MV vaccines. For this dissertation, I generated and performed preclinical evaluation of two novel MV vaccine candidates. Based on data from clinical trials that showed increasing the dosage of current MV vaccines improved antibody responses in six-month-old recipients, I hypothesized that increasing the relevant antigenic stimulus of a standard titer dose would allow safe and effective immunization at a younger age. I generated two modified MVs with increased expression of the hemagglutinin (H) protein, the most important viral antigen for inducing protective neutralizing immunity, in the background of a current vaccine-equivalent. One virus, MVvac2-H2, expressed higher levels of full-length H, resulting in a three-fold increase in H incorporation into virions, while the second, MVvac2-Hsol, expressed and secreted truncated, soluble H protein to its extracellular environment. The alteration to the virion envelope of MVvac2-H2 conferred upon that virus a measurable resistance to in vitro neutralization. In initial screening in adult mouse models of vaccination, both modified MVs proved more immunogenic than their parental strain in outbred mice, while MVvac2-H2 additionally proved more immunogenic in the gold standard MV-susceptible mouse model. Remarkably, MVvac2-H2 better induced protective immunity in the presence of low levels of artificially introduced passive immunity that mimic the passive maternal immunity that currently limits vaccination of young infants, and that strongly inhibited responses to the current vaccine-equivalent. Finally, I developed a more physiological infant-like mouse model for MV vaccine testing, in which MV-susceptible dams vaccinated with the current vaccine-equivalent transfer passive immunity to their pups. This model will allow additional preclinical evaluation of the performance of MVvac2-H2 in pups of immune dams. Altogether, in this dissertation I identify a promising candidate, MVvac2-H2, for a next generation measles vaccine.
ContributorsJulik, Emily (Author) / Reyes del Valle, Jorge (Thesis advisor) / Chang, Yung (Committee member) / Blattman, Joseph (Committee member) / Hogue, Brenda (Committee member) / Nickerson, Cheryl (Committee member) / Arizona State University (Publisher)
Created2016
Description
Host organisms have evolved multiple mechanisms to defend against a viral infection and likewise viruses have evolved multiple methods to subvert the host's anti-viral immune response. Vaccinia virus (VACV) is known to contain numerous proteins involved in blocking the cellular anti-viral immune response. The VACV E3L protein is important for inhibiting the anti-viral immune response and deletions within this gene lead to a severe attenuation. In particular, VACV containing N-terminal truncations in E3L are attenuated in animal models and fail to replicate in murine JC cells. Monkeypox virus (MPXV) F3L protein is a homologue of the VACV E3L protein, however it is predicted to contain a 37 amino acid N-terminal truncation. Despite containing an N-terminal truncation in the E3L homologue, MPXV is able to inhibit the anti-viral immune response similar to wild-type VACV and able to replicate in JC cells. This suggests that MPXV has evolved another mechanism(s) to counteract host defenses and promote replication in JC cells. MPXV produces less dsRNA than VACV during the course of an infection, which may explain why MPXV posses a phenotype similar to VACV, despite containing a truncated E3L homologue. The development of oncolytic viruses as a therapy for cancer has gained interest in recent years. Oncolytic viruses selectively replicate in and destroy cancerous cells and leave normal cells unharmed. Many tumors possess dysregulated anti-viral signaling pathways, since these pathways can also regulate cell growth. Creating a mutation in the N-terminus of the VACV-E3L protein generates an oncolytic VACV that depends on dysregulated anti-viral signaling pathways for replication allowing for direct targeting of the cancerous cells. VACV-E3Ldel54N selectively replicates in numerous cancer cells lines and not in the normal cell lines. Additionally, VACV-E3Ldel54N is safe and effective in causing tumor regression in a xenograph mouse model. Lastly, VACV-E3Ldel54N was capable of spreading from the treated tumors to the untreated tumors in both a xenograph and syngeneic mouse model. These data suggest that VACV-E3Ldel54N could be an effective oncolytic virus for the treatment of cancer.
ContributorsArndt, William D (Author) / Jacobs, Bertram (Thesis advisor) / Curtiss Iii, Roy (Committee member) / Chang, Yung (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2010
Description
The properties of adjuvants to stimulate an immune response to treat cancer has sparked a major area of research in the field of immunotherapy. Given the presence of multiple RNA sensors in mammalian host cells for eliciting innate immunity, synthetic RNA nanostructures present a unique opportunity for adjuvant exploration. While RNA nanostructures are organic and biocompatible in nature than other adjuvants, they could be tailored to have desired structural stability and functional diversity for in vivo application. In this study, a rectangular RNA origami nanostructure was designed to contain double-stranded RNA motifs and possess high structural stability. Using in vitro assays, RNA origami was shown to stimulate the toll-like receptor 3 (TLR3) signaling pathway, which has been reported to activate antigen presenting cells (APCs), natural killer (NK) cells, cluster of differentiation 8 (CD8) T-cells, and the secretion of proinflammatory cytokines. To explore RNA origami as an adjuvant for cancer immunotherapy, intraperitoneal administration of a murine colon cancer cell line (CT26) was used as a model system to mimic peritoneal metastasis (PM), in which RNA origami was investigated for its activities in mitigating PM tumor microenvironment and improving anti-tumor immunity. Given the poor outcome of the patients with PM and urgent need for new interventions, this study aims to translate the adjuvant activities of RNA origami demonstrated in vitro into potent anti-cancer immunotherapeutics. Here, it was shown that multiple intraperitoneal injections of RNA origami could inhibit tumor growth, leading to a significant delay and/or regression of metastatic tumor growth in the peritoneum. Furthermore, tumor-free mice, after being treated with RNA origami, were also resistant to a second challenge of tumor cells, indicating the development of the adaptive anti-tumor immunity. This immunity is dependent on T-cells since nude mice succumbed to tumor growth with or without RNA origami treatment. Thus, RNA-origami can function as an adjuvant to activate the innate immunity and subsequently the adaptive anti-tumor immunity, leading to tumor regression. Conceivably, RNA origami could be explored as an immunotherapeutic agent to improve the disease outcome of patients with peritoneal metastasis and peritoneal carcinogenesis.
ContributorsRodriguez del Villar, Ryan Luis (Author) / Chang, Yung (Thesis advisor) / Liu, Xiaowei (Committee member) / Qi, Xiaodong (Committee member) / Arizona State University (Publisher)
Created2018
Cancer autoantibody biomarker discovery and validation using nucleic acid programmable protein array
Description
Currently in the US, many patients with cancer do not benefit from the population-based screening, due to challenges associated with the existing cancer screening scheme. Blood-based diagnostic assays have the potential to detect diseases in a non-invasive way. Proteins released from small early tumors may only be present intermittently and get diluted to tiny concentrations in the blood, making them difficult to use as biomarkers. However, they can induce autoantibody (AAb) responses, which can amplify the signal and persist in the blood even if the antigen is gone. Circulating autoantibodies is a promising class of molecules that have potential to serve as early detection biomarkers for cancers. This Ph.D thesis aims to screen for autoantibody biomarkers for the early detection of two deadly cancer, basal-like breast cancer and lung adenocarcinoma. First, a method was developed to display proteins in both native and denatured conformation on protein array. This method adopted a novel protein tag technology, called HaloTag, to covalently immobilize proteins on glass slide surface. The covalent attachment allowed these proteins to endure harsh treatment without getting dissociated from slide surface, which enabled the profiling of antibody responses against both conformational and linear epitopes. Next, a plasma screening protocol was optimized to significantly increase signal to noise ratio of protein array based AAb detection. Following this, the AAb responses in basal-like breast cancer were explored using nucleic acid programmable protein arrays (NAPPA) containing 10,000 full-length human proteins in 45 cases and 45 controls. After verification in a large sample set (145 basal-like breast cancer cases / 145 controls / 70 non-basal breast cancer) by ELISA, a 13-AAb classifier was developed to differentiate patients from controls with a sensitivity of 33% at 98% specificity. Similar approach was also applied to the lung cancer study to identify AAbs that distinguished lung cancer patients from computed-tomography positive benign pulmonary nodules (137 lung cancer cases, 127 smoker controls, 170 benign controls). In this study, two panels of AAbs were discovered that showed promising sensitivity and specificity. Six out of eight AAb targets were also found to have elevated mRNA level in lung adenocarcinoma patients using TCGA data. These projects as a whole provide novel insights on the association between AAbs and cancer, as well as general B cell antigenicity against self-proteins.
ContributorsWang, Jie (Author) / LaBaer, Joshua (Thesis advisor) / Anderson, Karen S (Committee member) / Lake, Douglas F (Committee member) / Chang, Yung (Committee member) / Arizona State University (Publisher)
Created2015
Description
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen of Coronavirus Disease 2019 (COVID-19). Successful vaccination aims to elicit neutralizing antibodies (NAbs) which inhibit viral infection. Traditional NAb quantification methods (neutralization assays) are labor-intensive and expensive, with limited practicality for routine use (e.g. monitoring vaccination response). Thus, a rapid (10-minute) lateral flow assay (LFA) for quantification of SARS-CoV-2 NAbs was developed. Using the NAb LFA, an 18-month longitudinal study assessing monthly NAb titers was conducted in a cohort of over 500 COVID-19 mRNA vaccine recipients. Three NAb response groups were identified: vaccine strong responders (VSRs), moderate responders (VMRs), and poor responders (VPRs). VSRs generated high and durable NAb titers. VMRs initially generated high NAb titers but showed more rapid waning with time post-vaccination. Finally, VPRs rarely generated NAb titers ≥1:160, even after 3rd dose. Although strong humoral responses correlate with vaccine effectiveness, viral-specific CD4+ and CD8+ T cells are critical for long-term protection. Discordant phenotypes of viral-specific CD8+ and CD4+CXCR5+ T follicular helper (cTfh) cells have recently been associated with differential NAb responses. The second portion of this dissertation was to investigate whether/how SARS-CoV-2 T cell responses differ in individuals with impaired NAb titers following mRNA vaccination. Thus, phenotypic and functional characterization of T cell activation across NAb response groups was conducted. It was hypothesized that VPRs would exhibit discordant SARS-CoV-2 T cell activation and altered cTfh phenotypes. Peripheral blood mononuclear cells were isolated from VPRs, VMRs, VSRs, naturally infected, and normal donors. SARS-CoV-2 responsive T cells were characterized using in vitro activation induced marker assays, multicolor flow cytometry, and multiplex cytokine analysis. Further, CXCR5+ cTfh were examined for chemokine receptor expression (CCR6 and CXCR3). Results demonstrated that despite differential NAb responses, activation of SARS-CoV-2 responsive CD4+ and CD8+ T cells was comparable across NAb groups. However, double-positive CD4+CD8+, CD8low, and activated CD4+CXCR5+CCR6-CXCR3+ (Tfh1-like) T cells were expanded in VPRs compared to VMR and VSRs. Interestingly, a unique population of CD8+CXCR5+ T cells was also expanded in VPRs. These novel findings may aid in identification of individuals with impaired or altered immune responses to COVID-19 mRNA vaccination.
ContributorsRoeder, Alexa Jordan (Author) / Lake, Douglas (Thesis advisor) / McFadden, Grant (Committee member) / Borges Florsheim, Esther (Committee member) / Chang, Yung (Committee member) / Rahman, Masmudur (Committee member) / Arizona State University (Publisher)
Created2023