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- All Subjects: Cellular Biology
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
Cocaine abuse affects millions of people with disastrous medical and societal consequences. Despite this, there is still no FDA-approved treatment to decrease the likelihood of relapse in rehabilitated addicts, and acute cocaine toxicity (overdose) is only symptomatically treated. Studies have demonstrated a promising potential treatment option with the help of the human serum enzyme butyrylcholinesterase (BChE), an enzyme capable of breaking down cocaine into biologically inactive side products. This activity of wild-type BChE, however, is relatively low. This prompted the design of variants of BChE which exhibit significantly improved catalytic activity against cocaine. Plants were used as a sustainable, scalable, affordable platform system to produce large amounts of human biologics such as these cocaine hydrolase variants of BChE. Using a tobacco relative, Nicotiana benthamiana, recombinant enzymes can be produced at quantities relevant to clinical use with desired kinetic properties. Next, the ability of the most promising plant-produced cocaine super hydrolase, pCocSH, to counter the lethal effects of cocaine overdose in vivo was tested. These studies revealed that this plant-produced enzyme can protect mice from an otherwise lethal dose of cocaine. Most excitingly, it was found that pCocSH can rescue mice from overdose when given immediately after the onset of cocaine-induced seizures. These studies provide in vitro and in vivo proof-of-principle for a promising plant-derived biologic to be used as a pharmacokinetic-based treatment for cocaine addiction-related diseases such as overdose.
ContributorsLarrimore, Katherine E (Author) / Mor, Tsafrir S (Thesis advisor) / Gaxiola, Roberto (Committee member) / Mason, Hugh S (Committee member) / Neisewander, Janet L (Committee member) / Arizona State University (Publisher)
Created2015