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- All Subjects: Cellular Biology
- Creators: Chen, Qiang
Description
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) that emerged from a zoonotic host at the end of 2019 and caused a public health crisis. In this collection of studies, Nicotiana benthamiana plants are used to set the foundation for producing monoclonal antibodies (mAbs) with homogeneous glycosylation to neutralize SARS-CoV-2 and potentially address the immunopathology often observed with severe COVID-19. Specifically, a mAb against the human interleukin (IL)-6 receptor (sarilumab) was generated and evaluated in vitro for its potential to reduce IL-6 signaling that has been shown to be associated with more severe cases of COVID-19. Furthermore, multiple mAbs that bind to the receptor-binding domain (RBD) of SARS-CoV-2 and efficiently neutralize the virus were developed using plant-based expression. Several of these mAbs are from different classes of RBD-binding mAbs that have distinct binding sites from one another. Several mAbs from different classes showed synergy in neutralizing the ancestral strain of SARS-CoV-2 and a smaller subset showed synergy when tested against the highly mutated Omicron (B.1.1.529) variant. Of interest, a novel RBD-binding mAb, termed 11D7, that was raised against the ancestral strain and derived from a hybridoma, appears to have an epitope on the RBD that contributes more synergy to a mAb combination that efficiently neutralizes the B.1.1.529 variant of SARS-CoV-2. This epitope was partially mapped by competitive binding and shows that it overlaps with another known antibody that binds a cryptic, distal epitope, away from the receptor binding site, giving insight into the potential mechanism by which 11D7 neutralizes SARS-CoV-2, as well as potentially allowing it to resist SARS-CoV-2 immune evasion more efficiently. Furthermore, this mAb carries a highly homogeneous glycan pattern when expressed in N. benthamiana, that may contribute to enhanced effector function and provides a tool to elucidate the precise role of crystallizable fragment (Fc)-mediated protection in SARS-CoV-2 infection. Ultimately, these studies provide evidence of the utility of plant-made mAbs to be used as cocktail members, giving clarity to the use of less potent mAbs as valuable cocktail components which will spur further investigations into how mAbs with unique epitopes work together to efficiently neutralize SARS-CoV-2.
ContributorsJugler, Collin (Author) / Chen, Qiang (Thesis advisor) / Lake, Douglas (Committee member) / Steele, Kelly (Committee member) / Mason, Hugh (Committee member) / Arizona State University (Publisher)
Created2022
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
Environmental stressors can perturb cellular homeostasis. Cells activate an integrated stress response that will alleviate the effects of the ongoing stress. Stress-activated protein kinases function to phosphorylate the eukaryotic translation initiation factor, eIF2α, which results in inhibition of translation of house-keeping genes. Following these events, formation of cytoplasmic messenger ribonucleoprotein complexes, known as stress granules, will take place. Stress granules typically have a pro-survival function. These studies demonstrate that assembly of stress granules can also lead to necroptosis. Necroptosis is a caspase-independent, receptor-interacting protein kinase 3 (RIPK3)-dependent cell death pathway executed by mixed lineage kinase domain-like (MLKL) protein. Cellular stress is induced using arsenite (oxidative stress) or by infection with vaccinia virus (VACV) E3 protein Z-DNA-binding domain mutant, VACV-E3LΔ83N. In both cases, RIPK3-dependent death was observed in interferon (IFN)-primed L929 cells. This death led to phosphorylation and trimerization of MLKL, indicative of necroptosis. Necroptosis induced by oxidative stress and VACV-E3LΔ83N infection was dependent on the host Z-form nucleic acid sensor, DNA-dependent activator of IFN-regulatory factors (DAI), as it was inhibited in DAI-deficient L929 cells. Under both cellular stresses, DAI associated with RIPK3 and formed high-molecular-weight complexes, consistent with formation of the necrosomes. DAI localized into stress granules during necroptosis induced by arsenite and the mutant virus, and the necrosomes formed only in presence of stress granule assembly. The significance of stress granules for cellular stress-induced necroptosis was demonstrated using knock-out (KO) cell lines unable to form granules: T cell-restricted intracellular antigen 1 (TIA-1) KO MEF cells and Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1/2) KO U2OS cells. Necroptosis was inhibited in absence of stress granule formation as no cell death or activation of MLKL was observed in the knock-out cell lines following arsenite treatment or VACV-E3LΔ83N infection. Furthermore, wild-type VACV was able to inhibit stress granule assembly, which coincided with the virus ability to inhibit necroptosis. These studies have led to a model of Z-form nucleic acids being involved in activation of the stress granule-mediated necroptosis following induction by environmental stressors. These results have significance for understanding the etiology of human diseases and the antiviral innate immunity.
ContributorsSzczerba, Mateusz Bartlomiej (Author) / Jacobs, Bertram L (Thesis advisor) / Langland, Jeffrey (Committee member) / Lake, Douglas (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2021