Matching Items (5)
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- All Subjects: Molecular Biology
- All Subjects: Virus-like Particles
- Status: Published
Description
Malignant brain tumors are devastating despite aggressive treatments such as surgical resection, chemotherapy and radiation therapy. The average life expectancy of patients with newly diagnosed glioblastoma is approximately 15 months. One novel therapeutic strategy involves using a ketogenic diet (KD) which increases circulating ketones and reduces circulating glucose. While the preclinical work has shown that the KD increases survival, enhances radiation and alters several pathways in malignant gliomas, its impact on the anti-tumor immune response has yet to be examined. This dissertation demonstrates that mice fed the KD had increased tumor-reactive innate and adaptive immune responses, including increased cytokine production and cytolysis via tumor-reactive CD8+ T cells. Additionally, we saw that mice maintained on the KD had increased CD4 infiltration, while T regulatory cell numbers stayed consistent. Lastly, mice fed the KD had a significant reduction in immune inhibitory receptor expression as well as decreased inhibitory ligand expression on glioma cells, namely programmed death receptor -1 (PD-1) and its ligand programmed death receptor ligand -1 (PD-L1). Further, it is demonstrated that the ketone body beta-hydroxybutyrate (BHB) reduces expression of PD-L1 on glioma cells in vitro suggesting it may be responsible in part for immune-related changes elicited by the KD. Finally this dissertation also shows that the KD increases the expression of microRNAs predicted to target PD-L1 suggesting a potential mechanism to explain the ability of the KD to modulate immune inhibitory checkpoint pathways. Taken together these studies shed important light on the mechanisms underlying the KD and provide additional support for its use an adjuvant therapy for malignant glioma.
ContributorsWoolf, Eric Christopher (Author) / Compton, Carolyn C. (Thesis advisor) / Scheck, Adrienne C (Committee member) / Preul, Mark C (Committee member) / Blattman, Joseph N (Committee member) / Mehta, Shwetal (Committee member) / Arizona State University (Publisher)
Created2018
Description
Zika virus (ZIKV) outbreaks have been linked to several neurological pathologies in the developing fetus, which can progress to spontaneous abortion and microcephaly in newborns whose mothers were infected with the virus during pregnancy. ZIKV has also been correlated with neurological complications in adults such as Guillain-Barré Syndrome (GBS). ZIKV outbreaks often occur in low income areas with limited access to healthcare. Therefore, there is a need to create a low-cost preventative vaccine against the virus. Mature ZIKV particles contain a lipid bilayer, a positive sense single stranded RNA genome and three structural proteins: the envelope (E), membrane (M) and capsid (C) proteins. Congruently, to other members of the Flaviviridae family, ZIKV proteins are synthesized as a polyprotein precursor which needs to be processed to release the mature structural and non-structural viral proteins. Past studies have determined the ZIKV precursor protein is cleaved by a host furin protease which separates the Pr peptide and the M protein, while the host signal peptidase separates the M and E protein. Processing is important for correct folding of the E protein. In turn, the most important neutralizing antibodies upon infection are directed against epitopes of the E protein. In this work, we used a Bean Yellow Dwarf Viral vector system to transiently express, in Nicotiana benthamiana plants, a portion of the ZIKV polyprotein encoding the Pr, M and E proteins. I further demonstrate that plants can proteolytically process the polyprotein to yield the two integral membrane proteins M and E. These proteins can be shown to co-partition into a soluble membrane-particulate fraction, consistent with formation of enveloped virus-like particles (VLPs). This work provides the first step in creating a low-cost sustainable plant-based production system of ZIKV VLPs that can be explored as a potential component 0f a low-cost prophylactic vaccine against ZIKV.
ContributorsDi Palma, Michelle Pina (Author) / Mor, Tsafrir S (Thesis advisor) / Mason, Hugh S (Committee member) / Blattman, Joseph N (Committee member) / Arizona State University (Publisher)
Created2018
Description
Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E) protein as an immunogen to be vectored by a measles virus (MV) vaccine. However the domain III (DIII) of the dengue 2 E protein is too small to be immunogenic by itself. In order for it to be displayed on a larger particle, it was inserted into the amino terminus of small hepatitis B surface antigen (HBsAg, S) coding sequence. To generate the recombinant MV vector and verify the efficiency of this concept, a reverse genetics system was used where the MV vectors express one or two additional transcription units to direct the assembly of hybrid HBsAg particles. Two types of recombinant measles virus were produced: pB(+)MVvac2(DIII-S,S)P and pB(+)MVvac2(DIII-S)N. Virus recovered from pB(+)MVvac2(DIII-S,S)P was viable. An ELISA assay was performed to demonstrate the expression and secretion of HBsAg. Supernatant from MVvac2(DIII-S,S)P infected cells confirmed that hybrid HBsAg-domain III particles with a density similar to traditional HBsAg particles were released. Characteristics of the subviral particle have been analyzed for the successful incorporation of domain III. The replication fitness of the recombinant MV was evaluated using multi-step growth kinetics and showed reduced replication fitness when compared to the parental strain MVvac2. This demonstrates that viral replication is hindered by the addition of the two inserts into MV genome. Further analysis of MVvac2(DIII-S)N is needed to justify immune response studies in a small animal model using both of the generated recombinant vectors.
ContributorsHarahap, Indira Saridewi (Author) / Reyes del Valle, Jorge (Thesis director) / Hogue, Brenda (Committee member) / Misra, Rajeev (Committee member) / Barrett, The Honors College (Contributor) / T. Denny Sanford School of Social and Family Dynamics (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
The HIV-1 pandemic continues to cause millions of new infections and AIDS-related deaths each year, and a majority of these occur in regions of the world with limited access to antiretroviral therapy. Therefore, an HIV-1 vaccine is still desperately needed. The most successful HIV-1 clinical trial to date used a non-replicating canarypox viral vector and protein boosting, yet its modest efficacy left room for improvement. Efforts to derive novel vectors which can be both safe and immunogenic, have spawned a new era of live, viral vectors. One such vaccinia virus vector, NYVAC-KC, was specifically designed to replicate in humans and had several immune modulators deleted to improve immunogenicity and reduce pathogenicity. Two NYVAC-KC vectors were generated: one expressing the Gag capsid, and one with deconstructed-gp41 (dgp41), which contains an important neutralizing antibody target, the membrane proximal external region (MPER). These vectors were combined with HIV-1 Gag/dgp41 virus-like particles (VLPs) produced in the tobacco-relative Nicotiana benthamiana. Different plant expression vectors were compared in an effort to improve yield. A Geminivirus-based vector was shown to increase the amount of MPER present in VLPs, thus potentially enhancing immunogenicity. Furthermore, these VLPs were shown to interact with the innate immune system through Toll-like receptor (TLR) signaling, which activated antigen presenting cells to induce a Th2-biased response in a TLR-dependent manner. Furthermore, expression of Gag and dgp41 in NYVAC-KC vectors resulted in activation of antiviral signaling pathways reliant on TBK1/IRF3, which necessitated the use of higher doses in mice to match the immunogenicity of wild-type viral vectors. VLPs and NYVAC-KC vectors were tested in mice, ultimately showing that the best antibody and Gag-specific T cell responses were generated when both components were administered simultaneously. Thus, plant-produced VLPs and poxvirus vectors represent a highly immunogenic HIV-1 vaccine candidate that warrants further study.
ContributorsMeador, Lydia Rebecca (Author) / Mor, Tsafrir S (Thesis advisor) / Jacobs, Bertram L (Thesis advisor) / Blattman, Joseph N (Committee member) / Mason, Hugh S (Committee member) / Arizona State University (Publisher)
Created2016
Description
Plant-made virus-like particles (VLPs), composed of HIV-1 Gag and deconstructed gp41 proteins, have been shown to be safe and immunogenic in mice. Here, we report the successful production of HIV-1 Gag/dgp41 VLPs in Nicotiana benthamiana, using an enhanced geminivirus-based expression vector. This novel vector results in unique expression kinetics, with peak protein accumulation and minimal necrosis achieved on day 4 post-infiltration. In comparing various purification strategies, it was determined that a 20% ammonium sulfate precipitation is an effective and efficient method for removing plant proteins and purifying the recombinant VLPs of interest. If further purification is required, this may be achieved through ultracentrifugation. VLPs are a useful platform for a variety of biomedical applications and developing the technology to efficiently produce VLPs in the plant expression system is of critical importance.
ContributorsFleming, Claire (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Kamzina, Aigerim (Committee member) / Barrett, The Honors College (Contributor) / Department of Physics (Contributor) / School of Life Sciences (Contributor)
Created2022-05