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- All Subjects: Molecular Biology
- Creators: Chen, Qiang
- Creators: Misra, Rajeev
Description
Flavivirus infections are emerging as significant threats to human health around the globe. Among them West Nile(WNV) and Dengue Virus (DV) are the most prevalent in causing human disease with WNV outbreaks occurring in all areas around the world and DV epidemics in more than 100 countries. WNV is a neurotropic virus capable of causing meningitis and encephalitis in humans. Currently, there are no therapeutic treatments or vaccines available. The expanding epidemic of WNV demands studies that develop efficacious therapeutics and vaccines and produce them rapidly and inexpensively. In response, our lab developed a plant-derived monoclonal antibody (mAb) (pHu-E16) against DIII (WNV antigen) that is able to neutralize and prevent mice from lethal infection. However, this drug has a short window of efficacy due to pHu-E16's inability to cross the Blood Brain Barrier (BBB) and enter the brain. Here, we constructed a bifunctional diabody, which couples the neutralizing activity of E16 and BBB penetrating activity of 8D3 mAb. We also produced a plant-derived E16 scFv-CH1-3 variant with equivalent specific binding as the full pHu-E16 mAb, but only requiring one gene construct for production. Furthermore, a WNV vaccine based on plant-derived DIII was developed showing proper folding and potentially protective immune response in mice. DV causes severe hemorrhaging diseases especially in people exposed to secondary DV infection from a heterotypic strain. It is hypothesized that sub-neutralizing cross-reactive antibodies from the first exposure aid the second infection in a process called antibody-dependent enhancement (ADE). ADE depends on the ability of mAb to bind Fc receptors (FcγRs), and has become a major roadblock for developing mAb-based therapeutics against DV. We aim to produce an anti-Dengue mAb (E60) in different glycoengineered plant lines that exhibit reduced/differential binding to FcγRs, therefore, reducing or eliminating ADE. We have successfully cloned the molecular constructs of E60, and expressed it in two plant lines with different glycosylation patterns. We demonstrated that both plant-derived E60 mAb glycoforms retained specific recognition and neutralization activity against DV. Overall, our study demonstrates great strives to develop efficacious therapeutics and potent vaccine candidates against Flaviviruses in plant expression systems.
ContributorsHurtado, Jonathan (Author) / Chen, Qiang (Thesis advisor) / Huffman, Holly A (Committee member) / Steele, Kelly P (Committee member) / Arizona State University (Publisher)
Created2014
Description
V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy.
ContributorsWang, Guannan (Author) / Chang, Yung (Thesis advisor) / Levitus, Marcia (Committee member) / Misra, Rajeev (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2012
Description
Is it possible to treat the mouth as a natural environment, and determine new methods to keep the microbiome in check? The need for biodiversity in health may suggest that every species carries out a specific function that is required to maintain equilibrium and homeostasis within the oral cavity. Furthermore, the relationship between the microbiome and its host is mutually beneficial because the host is providing microbes with an environment in which they can flourish and, in turn, keep their host healthy. Reviewing examples of larger scale environmental shifts could provide a window by which scientists can make hypotheses. Certain medications and healthcare treatments have been proven to cause xerostomia. This disorder is characterized by a dry mouth, and known to be associated with a change in the composition, and reduction, of saliva. Two case studies performed by Bardow et al, and Leal et al, tested and studied the relationships of certain medications and confirmed their side effects on the salivary glands [2,3]. Their results confirmed a relationship between specific medicines, and the correlating complaints of xerostomia. In addition, Vissink et al conducted case studies that helped to further identify how radiotherapy causes hyposalivation of the salivary glands [4]. Specifically patients that have been diagnosed with oral cancer, and are treated by radiotherapy, have been diagnosed with xerostomia. As stated prior, studies have shown that patients having an ecologically balanced and diverse microbiome tend to have healthier mouths. The oral cavity is like any biome, consisting of commensalism within itself and mutualism with its host. Due to the decreased salivary output, caused by xerostomia, increased parasitic bacteria build up within the oral cavity thus causing dental disease. Every human body contains a personalized microbiome that is essential to maintaining health but capable of eliciting disease. The Human Oral Microbiomics Database (HOMD) is a set of reference 16S rRNA gene sequences. These are then used to define individual human oral taxa. By conducting metagenomic experiments at the molecular and cellular level, scientists can identify and label micro species that inhabit the mouth during parasitic outbreaks or a shifting of the microbiome. Because the HOMD is incomplete, so is our ability to cure, or prevent, oral disease. The purpose of the thesis is to research what is known about xerostomia and its effects on the complex microbiome of the oral cavity. It is important that researchers determine whether this particular perspective is worth considering. In addition, the goal is to create novel experiments for treatment and prevention of dental diseases.
ContributorsHalcomb, Michael Jordan (Author) / Chen, Qiang (Thesis director) / Steele, Kelly (Committee member) / Barrett, The Honors College (Contributor) / College of Letters and Sciences (Contributor)
Created2015-05
Description
Virus-Like Particles (VLPs) are self-assembling structures that lack the viral genetic material. Therefore they are safer and more immunogenic than other forms of vaccines. The Hepatitis B core (HBc) VLPs are a novel mechanism through which delivery of DNA-based human vaccines are plausible. Production of VLPs require recombinant, rapidly replicating, plant-based systems such as the geminiviral replicon system. This project entails the cloning process of HBc-DIII fusion protein, a VLP that should form Domain III of the Envelope protein on West Nile Virus, into deconstructed geminiviral vector. The cloning process includes the HBc-DIII fusion protein DNA isolation, restriction enzyme digestion with NcoI and SacI, PCR changing the NcoI site on the HBc-DIII insert to XbaI, sequencing, ligation into geminiviral vector and transformation into an agrobacterium strain. The major impediment to the cloning process was the presence of multiple bands instead of the expected two bands while doing restriction enzyme digests. The troubleshooting process enabled speculating that due to the excess of restriction enzymes in the digestion volume, some of the DNA was not digested completely. Hence, multiple bands were observed. However, sequencing analysis and further cloning process ensured the presence of HBc-DIII insert band (approximately 800bp) in the Gemini vector. Lastly, the construct HBc-DIII in Gemini vector was ensured to be in agrobacterium for further experiments such as agro-infiltration.
ContributorsSuresh Kumar, Reshma (Author) / Chen, Qiang (Thesis director) / Zhang, Peiming (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
The study of bacterial resistance to antimicrobial peptides (AMPs) is a significant area of interest as these peptides have the potential to be developed into alternative drug therapies to combat microbial pathogens. AMPs represent a class of host-mediated factors that function to prevent microbial infection of their host and serve as a first line of defense. To date, over 1,000 AMPs of various natures have been predicted or experimentally characterized. Their potent bactericidal activities and broad-based target repertoire make them a promising next-generation pharmaceutical therapy to combat bacterial pathogens. It is important to understand the molecular mechanisms, both genetic and physiological, that bacteria employ to circumvent the bactericidal activities of AMPs. These understandings will allow researchers to overcome challenges posed with the development of new drug therapies; as well as identify, at a fundamental level, how bacteria are able to adapt and survive within varied host environments. Here, results are presented from the first reported large scale, systematic screen in which the Keio collection of ~4,000 Escherichia coli deletion mutants were challenged against physiologically significant AMPs to identify genes required for resistance. Less than 3% of the total number of genes on the E. coli chromosome was determined to contribute to bacterial resistance to at least one AMP analyzed in the screen. Further, the screen implicated a single cellular component (enterobacterial common antigen, ECA) and a single transporter system (twin-arginine transporter, Tat) as being required for resistance to each AMP class. Using antimicrobial resistance as a tool to identify novel genetic mechanisms, subsequent analyses were able to identify a two-component system, CpxR/CpxA, as a global regulator in bacterial resistance to AMPs. Multiple previously characterized CpxR/A members, as well as members found in this study, were identified in the screen. Notably, CpxR/A was found to transcriptionally regulate the gene cluster responsible for the biosynthesis of the ECA. Thus, a novel genetic mechanism was uncovered that directly correlates with a physiologically significant cellular component that appears to globally contribute to bacterial resistance to AMPs.
ContributorsWeatherspoon-Griffin, Natasha (Author) / Shi, Yixin (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Misra, Rajeev (Committee member) / Nickerson, Cheryl (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2013
Description
Like most other phototrophic organisms the cyanobacterium Synechocystis sp. PCC 6803 produces carotenoids. These pigments often bind to proteins and assume various functions in light harvesting, protection from reactive oxygen species (ROS) and protein stabilization. One hypothesis was that carotenoids bind to the surface (S-)layer protein. In this work the Synechocystis S-layer protein was identified as Sll1951 and the effect on the carotenoid composition of this prokaryote by disruption of sll1951 was studied. Loss of the S-layer, which was demonstrated by electron microscopy, did not result in loss of carotenoids or changes in the carotenoid profile of the mutant, which was shown by HPLC and protein analysis. Although Δsll1951 was more susceptible to osmotic stress than the wild type, the general viability of the mutant remained unaffected. In a different study a combination of mutants having single or multiple deletions of putative carotenoid cleavage dioxygenase (CCD) genes was created. CCDs are presumed to play a role in the breakdown of carotenoids or apo-carotenoids. The carotenoid profiles of the mutants that were grown under conditions of increased reactive oxygen species were analyzed by HPLC. Pigment lifetimes of all strains were estimated by 13C-labeling. Carotenoid composition and metabolism were similar in all strains leading to the conclusion that the deleted CCDs do not affect carotenoid turnover in Synechocystis. The putative CCDs either do not fulfill this function in cyanobacteria or alternative pathways for carotenoid degradation exist. Finally, slr0941, a gene of unknown function but a conserved genome position in many cyanobacteria downstream of the δ-carotene desaturase, was disrupted. Initially, the mutant strain was impaired in growth but displayed a rather normal carotenoid content and composition, but an apparent second-site mutation occurred infrequently that restored growth rates and caused an accumulation of carotenoid isomers not found in the wild type. Based on the obtained data a role of the slr0941 gene in carotenoid binding/positioning for isomerization and further conversion to mature carotenoids is suggested.
ContributorsTrautner, Christoph (Author) / Vermaas, Willem Fj (Thesis advisor) / Chandler, Douglas E. (Committee member) / Misra, Rajeev (Committee member) / Bingham, Scott E (Committee member) / Arizona State University (Publisher)
Created2011
Description
Ebola hemorrhagic fever (EHF) is a severe and often fatal disease in human and nonhuman primates, caused by the Ebola virus. Approximately 30 years after the first epidemic, there is no vaccine or therapeutic medication approved to counter the Ebola virus. In this dissertation, a geminiviral replicon system was used to produce Ebola immune complex (EIC) in plant leaves and tested it as an Ebola vaccine. The EIC was produced in Nicotiana benthamiana leaves by fusing Ebola virus glycoprotein (GP1) to the C-terminus of heavy chain of 6D8 monoclonal antibody (mAb), which is specific to the 6D8 epitope of GP1, and co-expressing the fusion with the light chain of 6D8 mAb. EIC was purified by ammonium sulfate precipitation and protein A or protein G affinity chromatography. EIC was shown to be immunogenic in mice, but the level of antibody against Ebola virus was not sufficient to protect the mice from lethal the Ebola challenge. Hence, different adjuvants were tested in order to improve the immunogenicity of the EIC. Among several adjuvants that we used, Poly(I:C), which is a synthetic analog of double-stranded ribonucleic acid that can interact with a Toll-like receptor 3, strongly increased the efficacy of our Ebola vaccine. The mice immunized with EIC co-administered with Poly(I:C) produced high levels of neutralizing anti-Ebola IgG, and 80% of the mice were protected from the lethal Ebola virus challenge. Moreover, the EIC induced a predominant T-helper type 1 (Th1) response, whereas Poly(I:C) co-delivered with the EIC stimulated a mixed Th1/Th2 response. This result suggests that the protection against lethal Ebola challenge requires both Th1 and Th2 responses. In conclusion, this study demonstrated that the plant-produced EIC co-delivered with Poly(I:C) induced strong and protective immune responses to the Ebola virus in mice. These results support plant-produced EIC as a good vaccine candidate against the Ebola virus. It should be pursued further in primate studies, and eventually in clinical trials.
ContributorsPhoolcharoen, Waranyoo (Author) / Mason, Hugh S (Thesis advisor) / Chen, Qiang (Thesis advisor) / Arntzen, Charles J. (Committee member) / Change, Yung (Committee member) / Ma, Julian (Committee member) / Arizona State University (Publisher)
Created2010
Description
In this work, secretion of free fatty acids (FFAs) and ω-hydroxy FFAs wasachieved in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), and
FFAs were detected by a novel fluorescence assay. Current methods of detecting FFA
concentrations, including HPLC-based and GC-based methods or enzyme-based kits,
have hindered research advancement due to their laborious and/or expensive nature. The
work herein establishes a novel, rapid, fluorescence-based assay for detecting total FFA
concentrations secreted by Synechocystis FFA secretion strains. The novel FFA-detection
assay demonstrates the efficacy of using Nile Red as a fluorescent reporter for laurate or
palmitate at concentrations up to 500 µM in the presence of cationic surfactants. Total
FFA concentrations in Synechocystis supernatants quantified by the novel, Nile Red fluorescence-based assay are demonstrated herein to be highly correlative to total FFA
concentrations quantified by LC-MS; this correlation was seen in supernatant samples of
wild type Synechocystis and Synechocystis FFA secretion strains, both in 96-well plates
and 30-mL, aerated culture tubes.
This work also establishes the expression of a cytochrome P450 fusion enzyme,
CYP153A-CPRmut, or a monooxygenase system from Pseudomonas putida GPo1,
AlkBGT, in FFA secretion strains of Synechocystis for the generation of ω-hydroxy
laurate from laurate. After finding greatly increased ω-hydroxylation activity of
CYP153A-CPRmut with concurrent superoxide dismutase and catalase overexpression, 55
or 1.5 µM of ω-hydroxy laurate were produced over five days by Synechocystis strains
expressing CYP153A-CPRmut or AlkBGT, respectively. As further indication of the
presence of reactive oxygen species affecting ω-hydroxy laurate production with
Synechocystis strains expressing CYP153A-CPRmut, concentrations of ω-hydroxy laurate
in the supernatant increased over two-fold in the presence of 250 µM of the anti-oxidant,
methionine, in bench-scale cultures and in 96-well plate cultures. Additionally, a
mutation at the 55th amino acid position in AlkB (tryptophan to cysteine; AlkBW55C),
resulted in a more than two-fold shift in AlkB’s substrate preference from decanoate
towards the desired substrate, laurate. As a result, Synechocystis expressing AlkBW55C
could produce 5.9 µM ω-hydroxy laurate and 2.0 µM dodecanedioic acid over five days
of growth.
ContributorsAshe, Christopher (Author) / Vermaas, Willem Fj (Thesis advisor, Committee member) / Wang, Xuan (Committee member) / Nielsen, David R (Committee member) / Misra, Rajeev (Committee member) / Arizona State University (Publisher)
Created2023
Description
Vaccines are one of the most effective ways of combating infectious diseases and developing vaccine platforms that can be used to produce vaccines can greatly assist in combating global public health threats. This dissertation focuses on the development and pre-clinical testing of vaccine platforms that are highly immunogenic, easily modifiable, economically viable to produce, and stable. These criteria are met by the recombinant immune complex (RIC) universal vaccine platform when produced in plants. The RIC platform is modeled after naturally occurring immune complexes that form when an antibody, a component of the immune system that recognizes protein structures or sequences, binds to its specific antigen, a molecule that causes an immune response. In the RIC platform, a well-characterized antibody is linked via its heavy chain, to an antigen tagged with the antibody-specific epitope. The RIC antibody binds to the epitope tags on other RIC molecules and forms highly immunogenic complexes. My research has primarily focused on the optimization of the RIC platform. First, I altered the RIC platform to enable an N-terminal antigenic fusion instead of the previous C-terminal fusion strategy. This allowed the platform to be used with antigens that require an accessible N-terminus. A mouse immunization study with a model antigen showed that the fusion location, either N-terminal or C-terminal, did not impact the immune response. Next, I studied a synergistic response that was seen upon co-delivery of RIC with virus-like particles (VLP) and showed that the synergistic response could be produced with either N-terminal or C-terminal RIC co-delivered with VLP. Since RICs are inherently insoluble due to their ability to form complexes, I also examined ways to increase RIC solubility by characterizing a panel of modified RICs and antibody-fusions. The outcome was the identification of a modified RIC that had increased solubility while retaining high immunogenicity. Finally, I modified the RIC platform to contain multiple antigenic insertion sites and explored the use of bioinformatic tools to guide the design of a broadly protective vaccine.
ContributorsPardhe, Mary (Author) / Mason, Hugh S (Thesis advisor) / Chen, Qiang (Committee member) / Mor, Tsafrir (Committee member) / Wilson, Melissa (Committee member) / Arizona State University (Publisher)
Created2021
Description
Scientists are entrusted with developing novel molecular strategies for effective prophylactic and therapeutic interventions. Antivirals are indispensable tools that can be targeted at viral domains directly or at cellular domains indirectly to obstruct viral infections and reduce pathogenicity. Despite their transformative potential in healthcare, to date, antivirals have been clinically approved to treat only 10 out of the greater than 200 known pathogenic human viruses. Additionally, as obligate intracellular parasites, many virus functions are intimately coupled with host cellular processes. As such, the development of a clinically relevant antiviral is challenged by the limited number of clear targets per virus and necessitates an extensive insight into these molecular processes. Compounding this challenge, many viral pathogens have evolved to evade effective antivirals. Therefore, a means to develop virus- or strain-specific antivirals without detailed insight into each idiosyncratic biochemical mechanism may aid in the development of antivirals against a larger swath of pathogens. Such an approach will tremendously benefit from having the specific molecular recognition of viral species as the lowest barrier. Here, I modify a nanobody (anti-green fluorescent protein) that specifically recognizes non-essential epitopes (glycoprotein M-pHluorin chimera) presented on the extra virion surface of a virus (Pseudorabies virus strain 486). The nanobody switches from having no inhibitory properties (tested up to 50 μM) to ∼3 nM IC50 in in vitro infectivity assays using porcine kidney (PK15) cells. The nanobody modifications use highly reliable bioconjugation to a three-dimensional wireframe deoxyribonucleic acid (DNA) origami scaffold. Mechanistic studies suggest that inhibition is mediated by the DNA origami scaffold bound to the virus particle, which obstructs the internalization of the viruses into cells, and that inhibition is enhanced by avidity resulting from multivalent virus and scaffold interactions. The assembled nanostructures demonstrate negligible cytotoxicity (<10 nM) and sufficient stability, further supporting their therapeutic potential. If translatable to other viral species and epitopes, this approach may open a new strategy that leverages existing infrastructures – monoclonal antibody development, phage display, and in vitro evolution - for rapidly developing novel antivirals in vivo.
ContributorsPradhan, Swechchha (Author) / Hariadi, Rizal (Thesis advisor) / Hogue, Ian (Committee member) / Varsani, Arvind (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2022