Matching Items (11)
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- All Subjects: Plant Biology
- All Subjects: Dengue
- Creators: Mason, Hugh
- Creators: Chen, Qiang
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
Over the past decade, several high-value proteins have been produced using plant-based transient expression systems. However, these studies exposed some limitations that must be overcome to allow plant expression systems to reach their full potential. These limitations are the low level of recombinant protein accumulation achieved in some cases, and lack of efficient co-expression vectors for the production of multi-protein complexes. This study report that tobacco Extensin (Ext) gene 3' untranslated region (UTR) can be broadly used to enhance recombinant protein expression in plants. Extensin is the hydroxyproline-rich glycoprotein that constitutes the major protein component of cell walls. Using transient expression, it was found that the Ext 3' UTR increases recombinant protein expression up to 13.5- and 6-fold in non-replicating and replicating vector systems, respectively, compared to previously established terminators. Enhanced protein accumulation was correlated with increased mRNA levels associated with reduction in read-through transcription. Regions of Ext 3' UTR essential for maximum gene expression included a poly-purine sequence used as a major poly-adenylation site. Furthermore, modified bean yellow dwarf virus (BeYDV)-based vectors designed to allow co-expression of multiple recombinant genes were constructed and tested for their performance in driving transient expression in plants. Robust co-expression and assembly of heavy and light chains of the anti-Ebola virus monoclonal antibody 6D8, as well as E. coli heat-labile toxin (LT) were achieved with the modified vectors. The simultaneous co-expression of three fluoroproteins using the single replicon, triple cassette is demonstrated by confocal microscopy. In conclusion, this study provides an excellent tool for rapid, cost-effective, large-scale manufacturing of recombinant proteins for use in medicine and industry.
ContributorsRosenthal, Sun Hee (Author) / Mason, Hugh (Thesis advisor) / Mor, Tsafrir (Committee member) / Chang, Yung (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2012
Description
Variants of human butyrylcholinesterase (BChE) have been designed to have high cocaine hydrolytic activity. These variants have potential pharmacological applications toward treating cocaine overdose and addiction. These enzymes must be stable in the human body over fairly long periods of time in order to be effective at treating cocaine addiction. Recombinantly expressed BChE, however, tends to be in monomer or dimer oligomeric forms, which are far less stable than the tetramer form of the enzyme. When BChE is transiently expressed in Nicotiana benthamiana, it is produced mainly as monomers and dimers. However, when the protein is expressed through stable transformation, it produces much greater proportions of tetramers. Tetramerization of WT human plasma derived BChE is facilitated by the binding of a proline rich peptide. In this thesis, I investigated if a putative plant-derived analog of the mammalian proline-rich attachment domain caused stably expressed cocaine hydrolase variants of human BChE to undergo tetramerization. I also examined if co-expression of peptides with known proline-rich attachment domains further shifted the monomer-tetramer ratio toward the tetramer.
ContributorsKendle, Robert Player (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Larrimore, Kathy (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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
Influenza is a deadly disease for which effective vaccines are sorely lacking. This is largely due to the phenomena of antigenic shift and drift in the influenza virus's surface proteins, hemagglutinin (HA) and neuraminidase (NA). The ectodomain of the matrix 2 protein (M2e) of influenza A, however, has demonstrated high levels of conservation. On its own it is poorly immunogenic and offers little protection against influenza infections, but by combining it with a potent adjuvant, this limitation may be overcome. Recombinant immune complexes, or antigens fused to antibodies that have been engineered to form incredibly immunogenic complexes with one another, were previously shown to be useful, immunogenic platforms for the presentation of various antigens and could provide the boost in immunogenicity that M2e needs to become a powerful universal influenza A vaccine. In this thesis, genetic constructs containing geminiviral replication proteins and coding for a consensus sequence of dimeric M2e fused to antibodies featuring complimentary epitopes and epitope tags were generated and used to transform Agrobacterium tumefaciens. The transformed bacteria was then used to cause Nicotiana benthamiana to transiently express M2e-RICs at very high levels, with enough RICs being gathered to evaluate their potency in future mouse trials. Future directions and areas for further research are discussed.
ContributorsFavre, Brandon Chetan (Author) / Mason, Hugh (Thesis director) / Mor, Tsafrir (Committee member) / Diamos, Andrew (Committee member) / Department of Psychology (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Vaccines against the arthropod-borne dengue virus (DENV) are still commercially nonexistent. A subunit immunization strategy may be of value, especially if a safe viral vector acts as a biologically active adjuvant. The DENV envelope protein (E), the main target for neutralizing immune responses, has three conformational domains. The immunoglobulin-like and independently folding domain III (DIII) contains epitopes that elicit highly specific neutralizing antibodies. The hepatitis B small surface antigen (HBsAg, S) was used as a scaffold to display DENV 2 DIII on a virus-like particle (VLP). A measles virus (MV) was engineered to vector HBsAg and the hybrid glycoprotein DIII-HBsAg in two different loci (DIII-S). Despite the relatively deleterious effect on replication caused by the insertion of two transcription cassettes, the recombinant virus MVvac2(DIII-S,S)P induced the secretion of DIII-S hybrid VLP with a similar sucrose density as HBsAg particles (1.10-1.12g/ml) and peaked at 48 h post-infection producing 1.3x106 TCID50/ml infectious MV units in vitro. A second recombinant virus, MVvac2(DIII-S)N, was engineered to vector only the hybrid DIII-S. However, it did not induce the secretion of hybrid HBsAg particles in the supernatant of infected cells. The immunogenicity of the recombinant viruses was tested in a MV-susceptible small animal model, the experimental group which received two 105 TCID50 I.P. doses of MVvac2(DIII-S,S)P in a 28 day interval developed a robust immune response against MV (1:1280), HBsAg (787 mIU/ml) and DENV2 (Log10 neutralization index of 1.2) on average. In summary, it is possible to display DENV E DIII on hybrid HBsAg particles vectored by MV that elicit an immune response. This forms the basis for a potential vaccine platform against DENV.
ContributorsHarahap, Indira (Author) / Reyes del Valle, Jorge (Thesis advisor) / Hogue, Brenda G (Thesis advisor) / Lake, Douglas (Committee member) / Mason, Hugh (Committee member) / Arizona State University (Publisher)
Created2015
Description
Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age, whom are particularly prone to Dengue severe infection and death, cannot be immunized using current approved DV vaccine. The most important issues documented so far are the lack of efficiency and enhancement of the disease in young seronegative recipients, as well as uneven protection against the four DV serotypes. Based on data from clinical trials that showed enhanced performance of dengue vaccines when the host has previous anti-flaviviral immunity, I proposed here an attractive solution to complement the current vaccine: a recombinant measles vaccine vectoring dengue protective antigens to be administered to young infants. I hypothesized that recombinant measles virus expressing Dengue 2 and 4 antigens would successfully induce neutralizing responses against DV2 and 4 and the vaccine cocktail of this recombinant measles can prime anti-flaviviral neutralizing immunity. For this dissertation, I generated and performed preclinical immune assessment for four novel Measles-Dengue (MV-DV) vaccine candidates. I generated four MVs expressing the pre membrane (prM) and full length or truncated (90%) forms of the major envelope (E) from DV2 and DV4. Two virus, MVvac2-DV2(prME)N and MVvac2-DV4(prME), expressed high levels of membrane associated full-length E, while the other two viruses, MVvac2-DV2(prMEsol)N and MVvac2-DV4(prMEsol)N, expressed and secreted truncated, soluble E protein to its extracellular environment. The last two vectored vaccines proved superior anti-dengue neutralizing responses comparing to its corresponding full length vectors. Remarkably, when MVvac2-DV2/4(prMEsol)N recombinant vaccines were combined, the vaccine cocktail was able to prime cross-neutralizing responses against DV 1 and the relatively distant 17D yellow fever virus attenuated strain. Thus, I identify a promising DV vaccination strategy, MVvac2-DV2/4(prMEsol)N, which can prime broad neutralizing immune responses by using only two of the four available DV serotypes. The current MV immunization scheme can be advantageus to prime broad anti-flaviviral neutralizing immunity status, which will be majorly boosted by subsequent chimeric Dengue vaccine approaches.
ContributorsAbdelgalel, Rowida (Author) / Reyes del Valle, Jorge (Thesis advisor) / Mason, Hugh (Thesis advisor) / Lake, Douglas (Committee member) / Stout, Valerie (Committee member) / Frasch, Wayne (Committee member) / Arizona State University (Publisher)
Created2016
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
Flaviviruses (FVs) are among the most medically important arboviruses of the world with the Dengue virus (DENV) accounting for a large percentage of infections observed in tropical and subtropical regions of the world. Globalization, travel, and the expanding range of mosquito vectors, such as Aedes aegypti, have increased the potential of infection rates and illnesses associated with FVs.
The DENV and the Zika (ZIKV) FVs frequently co-circulate and generally cause mild self-liming febrile illnesses. However, a secondary infection with a heterologous DENV serotype may lead to life threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF/DSS have been linked to antibody dependent enhancement of infection (ADE), a phenomenon that occurs when antibodies (Abs) formed against an initial infection with one serotype of DENV cross-reacts but does not neutralize a heterologous DENV serotype in a secondary infection. Furthermore, Abs raised against the ZIKV have been observed to cross-react with the DENV and vice versa, which can potentially cause ADE and lead to severe DENV disease. The ZIKV can be transmitted vertically and has been linked to devastating congenital defects such as microcephaly in newborns. FDA approved treatments do not exist for DENV and ZIKV illnesses. Thus, there is a need for safe and effective treatments for these co-circulating viruses. Here, a tetravalent bispecific antibody (bsAb) targeting the ZIKV and all four serotypes of the DENV was expressed in the Nicotiana benthamiana (N. benthamiana) plant. Functional assays of the DENV/ZIKV bsAb demonstrated binding, neutralization, and a significant reduction in ADE activity against both the DENV and the ZIKV.
A single chain variable fragment (scFv) and a diabody based on an antibody directed against the immune checkpoint inhibitor PD-L1, were also expressed in N. benthamiana leaves. The smaller sizes of the scFv and diabody confers them with the ability to penetrate deeper tissues making them beneficial in diagnostics, imaging, and possibly cancer therapy. The past few decades has seen long strives in recombinant protein production in plants with significant improvements in production, safety, and efficacy. These characteristics make plants an attractive platform for the production of recombinant proteins, biologics, and therapeutics.
The DENV and the Zika (ZIKV) FVs frequently co-circulate and generally cause mild self-liming febrile illnesses. However, a secondary infection with a heterologous DENV serotype may lead to life threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF/DSS have been linked to antibody dependent enhancement of infection (ADE), a phenomenon that occurs when antibodies (Abs) formed against an initial infection with one serotype of DENV cross-reacts but does not neutralize a heterologous DENV serotype in a secondary infection. Furthermore, Abs raised against the ZIKV have been observed to cross-react with the DENV and vice versa, which can potentially cause ADE and lead to severe DENV disease. The ZIKV can be transmitted vertically and has been linked to devastating congenital defects such as microcephaly in newborns. FDA approved treatments do not exist for DENV and ZIKV illnesses. Thus, there is a need for safe and effective treatments for these co-circulating viruses. Here, a tetravalent bispecific antibody (bsAb) targeting the ZIKV and all four serotypes of the DENV was expressed in the Nicotiana benthamiana (N. benthamiana) plant. Functional assays of the DENV/ZIKV bsAb demonstrated binding, neutralization, and a significant reduction in ADE activity against both the DENV and the ZIKV.
A single chain variable fragment (scFv) and a diabody based on an antibody directed against the immune checkpoint inhibitor PD-L1, were also expressed in N. benthamiana leaves. The smaller sizes of the scFv and diabody confers them with the ability to penetrate deeper tissues making them beneficial in diagnostics, imaging, and possibly cancer therapy. The past few decades has seen long strives in recombinant protein production in plants with significant improvements in production, safety, and efficacy. These characteristics make plants an attractive platform for the production of recombinant proteins, biologics, and therapeutics.
ContributorsEsqueda, Adrian (Author) / Chen, Qiang (Thesis advisor) / Arntzen, Charles (Committee member) / Lake, Douglas (Committee member) / Mason, Hugh (Committee member) / Arizona State University (Publisher)
Created2019
Description
Plant viral vectors have previously been used to produce high expression levels of antibodies and other proteins of interest. By utilizing a transformed Agrobacterium with the vector containing the protein of interest for infiltration, viral vectors can easily reach the plant cells making it an effective form of transient protein expression. For this project two different plant viral vectors were compared; the geminiviral vector derived from Bean yellow dwarf virus (BeYDV) and the MagnICON vector system derived from Tobacco Mosaic Virus(TMV) and Potato Virus X(PVX). E16, an antibody against West Nile virus, has previously been expressed using both systems but expression levels between the systems were not directly compared. Agrobacterium tumefaciens EHA105 cells were transformed with both systems and expression levels of E16 were quantified using ELISAs. Results showed very low expression levels of E16 using the geminiviral vector indicating a need for further investigation into the clone used as previous studies reported much higher expression levels with the system.
ContributorsMurphy, Skylar (Author) / Chen, Qiang (Thesis director) / Jugler, Collin (Committee member) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05