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- Genre: Doctoral Dissertation
Biological and immunological characterization of plant-produced HIV-1 Gag/dgp41 virus-like particles
Description
Anti-retroviral drugs and AIDS prevention programs have helped to decrease the rate of new HIV-1 infections in some communities, however, a prophylactic vaccine is still needed to control the epidemic world-wide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although recent clinical trials have shown promising results. Recent successes have focused on highly conserved, mucosally-targeted antigens within HIV-1 such as the membrane proximal external region (MPER) of the envelope protein, gp41. MPER has been shown to play critical roles in the viral mucosal transmission, though this peptide is not immunogenic on its own. Gag is a structural protein configuring the enveloped virus particles, and has been suggested to constitute a target of the cellular immunity potentially controlling the viral load. It was hypothesized that HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (dgp41) could be expressed in plants. Plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a tobacco mosaic virus-based expression system or a combination of both. Results of biophysical, biochemical and electron microscopy characterization demonstrated that plant cells could support not only the formation of HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These particles were purified and utilized in mice immunization experiments. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR - a fusion of MPER and the B-subunit of cholera toxin) were administered to BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens could be elicited in mice systemically primed with VLPs and these responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a robust boosting response against Gag and gp41 when boosted with either candidate. Functional assays of these antibodies are in progress to test the antibodies' effectiveness in neutralizing and preventing mucosal transmission of HIV-1. This immunogenicity of plant-based Gag/dgp41 VLPs represents an important milestone on the road towards a broadly-efficacious and inexpensive subunit vaccine against HIV-1.
ContributorsKessans, Sarah (Author) / Mor, Tsafrir S (Thesis advisor) / Matoba, Nobuyuki (Committee member) / Mason, Hugh (Committee member) / Hogue, Brenda (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Description
The concept of vaccination dates back further than Edward Jenner's first vaccine using cowpox pustules to confer immunity against smallpox in 1796. Nevertheless, it was Jenner's success that gave vaccines their name and made vaccinia virus (VACV) of particular interest. More than 200 years later there is still the need to understand vaccination from vaccine design to prediction of vaccine efficacy using mathematical models. Post-exposure vaccination with VACV has been suggested to be effective if administered within four days of smallpox exposure although this has not been definitively studied in humans. The first and second chapters analyze post-exposure prophylaxis of VACV in an animal model using v50ΔB13RMγ, a recombinant VACV expressing murine interferon gamma (IFN-γ) also known as type II IFN. While untreated animals infected with wild type VACV die by 10 days post-infection (dpi), animals treated with v50ΔB13RMγ 1 dpi had decreased morbidity and 100% survival. Despite these differences, the viral load was similar in both groups suggesting that v50ΔB13RMγ acts as an immunoregulator rather than as an antiviral. One of the main characteristics of VACV is its resistance to type I IFN, an effect primarily mediated by the E3L protein, which has a Z-DNA binding domain and a double-stranded RNA (dsRNA) binding domain. In the third chapter a VACV that independently expresses both domains of E3L was engineered and compared to wild type in cells in culture. The dual expression virus was unable to replicate in the JC murine cell line where both domains are needed together for replication. Moreover, phosphorylation of the dsRNA dependent protein kinase (PKR) was observed at late times post-infection which indicates that both domains need to be linked together in order to block the IFN response. Because smallpox has already been eradicated, the utility of mathematical modeling as a tool for predicting disease spread and vaccine efficacy was explored in the last chapter using dengue as a disease model. Current modeling approaches were reviewed and the 2000-2001 dengue outbreak in a Peruvian region was analyzed. This last section highlights the importance of interdisciplinary collaboration and how it benefits research on infectious diseases.
ContributorsHolechek, Susan A (Author) / Jacobs, Bertram L (Thesis advisor) / Castillo-Chavez, Carlos (Committee member) / Frasch, Wayne (Committee member) / Hogue, Brenda (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2011
Description
Vaccinia virus (VACV) is the current vaccine for the highly infectious smallpox disease. Since the eradication of smallpox, VACV has been developed extensively as a heterologous vaccine vector for several pathogens. However, due to the complications associated with this replication competent virus, the safety and efficacy of VACV vaccine vector has been reevaluated. To evaluate the safety and efficacy of VACV, we study the interactions between VACV and the host innate immune system, especially the type I interferon (IFN) signaling pathways. In this work, we evaluated the role of protein kinase R (PKR) and Adenosine Deaminase Acting on RNA 1(ADAR1), which are induced by IFN, in VACV infection. We found that PKR is necessary but is not sufficient to activate interferon regulatory factor 3 (IRF3) in the induction of type I IFN; and the activation of the stress-activated protein kinase/ c-Jun NH2-terminal kinase is required for the PKR-dependent activation of IRF3 during VACV infection. Even though PKR was found to have an antiviral effect in VACV, ADAR1 was found to have a pro-viral effect by destabilizing double stranded RNA (dsRNA), rescuing VACVΔE3L, VACV deleted of the virulence factor E3L, when provided in trans. With the lessons we learned from VACV and host cells interaction, we have developed and evaluated a safe replication-competent VACV vaccine vector for HIV. Our preliminary results indicate that our VACV vaccine vector can still induce the IFN pathway while maintaining the ability to replicate and to express the HIV antigen efficiently. This suggests that this VACV vector can be used as a safe and efficient vaccine vector for HIV.
ContributorsHuynh, Trung Phuoc (Author) / Jacobs, Bertram L (Thesis advisor) / Hogue, Brenda (Committee member) / Chang, Yung (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2013
Description
Coronaviruses are medically important viruses that cause respiratory and enteric infections in humans and animals. The recent emergence through interspecies transmission of severe acute respiratory syndrome coronavirus (SARS-CoV) strongly supports the need for development of vaccines and antiviral reagents. Understanding the molecular details of virus assembly is an attractive target for development of such therapeutics. Coronavirus membrane (M) proteins constitute the bulk of the viral envelope and play key roles in assembly, through M-M, M-spike (S) and M-nucleocapsid (N) interactions. M proteins have three transmembrane domains, flanked by a short amino-terminal domain and a long carboxy-terminal tail located outside and inside the virions, respectively. Two domains are apparent in the long tail - a conserved region (CD) at the amino end and a hydrophilic, charged carboxy-terminus (HD). We hypothesized that both domains play functionally important roles during assembly. A series of changes were introduced in the domains and the functional impacts were studied in the context of the virus and during virus-like particle (VLP) assembly. Positive charges in the CD gave rise to viruses with neutral residue replacements that exhibited a wild-type phenotype. Expression of the mutant proteins showed that neutral, but not positive, charges formed VLPs and coexpression with N increased output. Alanine substitutions resulted in viruses with crippled phenotypes and proteins that failed to assemble VLPs or to be rescued into the envelope. These viruses had partially compensating changes in M. Changes in the HD identified a cluster of three key positive charges. Viruses could not be recovered with negatively charged amino acid substitutions at two of the positions. While viruses were recovered with a negative charge substitution at one of the positions, these exhibited a severely crippled phenotype. Crippled mutants displayed a reduction in infectivity. Results overall provide new insight into the importance of the M tail in virus assembly. The CD is involved in fundamental M-M interactions required for envelope formation. These interactions appear to be stabilized through interactions with the N protein. Positive charges in the HD also play an important role in assembly of infectious particles.
ContributorsArndt, Ariel L (Author) / Hogue, Brenda G (Thesis advisor) / Jacobs, Bertram (Committee member) / Francisco, Wilson (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2010
Description
Macrophage fusion resulting multinucleated giant cells (MGCs) formation is associated with numerous chronic inflammatory diseases including the foreign body reaction to implanted
biomaterials. Despite long-standing predictions, there have been attempts to use live-cell
imaging to investigate the morphological features initiating macrophage fusion because
macrophages do not fuse on clean glass required for most imaging techniques. Consequently,
the mechanisms of macrophage fusion remain poorly understood. The goal of this research
project was to characterize the early and late stages of macrophage multinucleation using
fusogenic optical quality substrate. Live-cell imaging with phase-contrast and lattice-light
sheet microscopy revealed that an actin-based protrusion initiates macrophage fusion. WASpdeficient macrophages and macrophages isolated from myeloid cell-specific Cdc42-/- mice
fused at very low rates. In addition, inhibiting the Arp2/3 complex impaired both the formation
of podosomes and macrophage fusion.
Analyses of the late stages of macrophage multinucleation on biomaterials implanted into
mice revealed novel actin-based zipper-like structures (ZLSs) formed at contact sites between
MGCs. The model system that was developed for the induction of ZLSs in vitro allowed for
the characterization of protein composition using confocal and super-resolution microscopy.
Live-cell imaging demonstrated that ZLSs are dynamic formations undergoing continuous
assembly and disassembly and that podosomes are precursors of these structures. It was further
found that E-cadherin and nectin-2 are involved in ZLS formation by bridging the plasma
membranes together. ii
Macrophage fusion on implanted biomaterials inherently involves their adhesion to the
implant surface. While biomaterials rapidly acquire a layer of host proteins, a biological
substrate that is required for macrophage fusion is unknown. It was shown that mice with
fibrinogen deficiency as well as mice expressing fibrinogen incapable of fibrin polymerization
displayed a dramatic reduction of macrophage fusion on biomaterials. Furthermore, these mice
were protected from the formation of the dense collagenous capsule enveloping the implant. It
was also found that the main cell type responsible for the deposition of collagen in the capsule
were mononuclear macrophages but not myofibroblasts. Together, these findings reveal a
critical role of the actin cytoskeleton in macrophage fusion and identify potential targets to
reduce the drawbacks of macrophage fusion on implanted biomaterials.
ContributorsBalabiyev, Arnat (Author) / Ugarova, Tatiana (Thesis advisor) / Roberson, Robert (Committee member) / Chandler, Douglas (Committee member) / Baluch, Page (Committee member) / Arizona State University (Publisher)
Created2021
Description
Macromolecular structural biology advances the understanding of protein function through the structure-function relationship for applications to scientific challenges like energy and medicine. The proteins described in these studies have applications to medicine as targets for therapeutic drug design. By understanding the mechanisms and dynamics of these proteins, therapeutics can be designed and optimized based on their unique structural characteristics. This can create new, focused therapeutics for the treatment of diseases with increased specificity — which translates to greater efficacy and fewer off-target effects. Many of the structures generated for this purpose are “static” in nature, meaning the protein is observed like a still-frame photograph; however, the use of time-resolved techniques is allowing for greater understanding of the dynamic and flexible nature of proteins. This work advances understanding the dynamics of the medically relevant proteins NendoU and Taspase1 using serial crystallography to establish conditions for time-resolved, mix-and-inject crystallographic studies.
ContributorsJernigan, Rebecca Jeanne (Author) / Fromme, Petra (Thesis advisor) / Hansen, Debra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Hogue, Brenda (Committee member) / Arizona State University (Publisher)
Created2022
Description
Poxviruses such as monkeypox virus (MPXV) are emerging zoonotic diseases. Compared to MPXV, Vaccinia virus (VACV) has reduced pathogenicity in humans and can be used as a partially protective vaccine against MPXV. While most orthopoxviruses have E3 protein homologues with highly similar N-termini, the MPXV homologue, F3, has a start codon mutation leading to an N-terminal truncation of 37 amino acids. The VACV protein E3 consists of a dsRNA binding domain in its C-terminus which must be intact for pathogenicity in murine models and replication in cultured cells. The N-terminus of E3 contains a Z-form nucleic acid (ZNA) binding domain and is also required for pathogenicity in murine models. Poxviruses produce RNA transcripts that extend beyond the transcribed gene which can form double-stranded RNA (dsRNA). The innate immune system easily recognizes dsRNA through proteins such as protein kinase R (PKR). After comparing a vaccinia virus with a wild-type E3 protein (VACV WT) to one with an E3 N-terminal truncation of 37 amino acids (VACV E3Δ37N), phenotypic differences appeared in several cell lines. In HeLa cells and certain murine embryonic fibroblasts (MEFs), dsRNA recognition pathways such as PKR become activated during VACV E3Δ37N infections, unlike VACV WT. However, MPXV does not activate PKR in HeLa or MEF cells. Additional investigation determined that MPXV produces less dsRNA than VACV. VACV E3Δ37N was made more similar to MPXV by selecting mutants that produce less dsRNA. By producing less dsRNA, VACV E3Δ37N no longer activated PKR in HeLa or MEF cells, thus restoring the wild-type phenotype. Furthermore, in other cell lines such as L929 (also a murine fibroblast) VACV E3Δ37N, but not VACV WT infection leads to activation of DNA-dependent activator of IFN-regulatory factors (DAI) and induction of necroptotic cell death. The same low dsRNA mutants demonstrate that DAI activation and necroptotic induction is independent of classical dsRNA. Finally, investigations of spread in an animal model and replication in cell lines where both the PKR and DAI pathways are intact determined that inhibition of both pathways is required for VACV E3Δ37N to replicate.
ContributorsCotsmire, Samantha (Author) / Jacobs, Bertram L (Thesis advisor) / Varsani, Arvind (Committee member) / Hogue, Brenda (Committee member) / Haydel, Shelley (Committee member) / Arizona State University (Publisher)
Created2021
Description
Mucosal membranes represent a major site of pathogen transmission and cancer development. Enhancing T cell migration to mucosal surfaces could improve immune-based therapies for these diseases, yielding better clinical outcomes. All-trans-retinoic acid (ATRA) is a biologically active form of vitamin A that has been shown to increase T cell migration to mucosal sites, however its therapeutic use is limited by its toxicity potential and unstable nature. ATRA-related compounds with lower toxicity and higher stability were assessed for their ability to induce similar immune migration effects as ATRA, using in vitro and in vivo model systems. Chapter 2 summarizes the first project, in which synthetic, ATRA-like compounds called rexinoids were used to modulate T cell expression of mucosal homing proteins chemokine receptor 9 (CCR9) and integrin alpha 4 beta 7 (α4β7), and alter their physical migration in vitro. Several rexinoids independently mimicked the activity of ATRA to enhance protein expression and migration, while others worked synergistically with subtoxic doses of ATRA to produce similar results. Furthermore, rexinoid administration in vivo was well-tolerated by animal models, a finding not seen with ATRA.
Chapter 3 focuses on the second project, where plasmids containing ATRA-synthesizing proteins were assessed for their in vivo ability to act as mucosal vaccine adjuvants and enhance T cell migration to mucosal sites during DNA vaccination. Though increased mucosal migration was seen with use of the adjuvant plasmids, these findings were not determined to be significant. Immune-mediated protection following viral challenge was also not determined to be significant in animal models receiving both vaccine and adjuvant plasmids.
The data shows that several novel rexinoids may possess enhanced clinical utility compared to ATRA, lending support for their use in immunotherapeutic approaches towards mucosal maladies. While the potential mucosal vaccine adjuvants did not show great significance in enhancing T cell migration or viral protection, further optimization of the model system may produce better results. This work helps advance knowledge of immune cell trafficking to afflicted mucosal regions. It can be used as a basis for understanding migration to other body areas, as well as for the development of better immune-based treatments.
ContributorsManhas, Kavita Rani (Author) / Blattman, Joseph (Thesis advisor) / Marshall, Pamela (Committee member) / Lake, Douglas (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2022
Description
Alpha herpesviruses are a family of neuroinvasive viruses that infect multiplevertebrate species. Alpha herpesviruses are responsible for human and livestock infections, most notably Herpes Simplex Virus (HSV), Varicella Zoster virus (VZV), and Pseudorabies Virus (PRV). PRV is a potent swine virus that can infect other mammals, and results in lethal encephalitis that can be devastating to livestock and of great financial expense to farmers. HSV, types 1 and 2, and VZV are widespread throughout the global human population, with estimates of the HSV-1 burden at about 60% of people worldwide. The hallmark of alpha herpesvirus infection is a persistent, lifelong infection that can reactivate throughout the lifespan of the host. Currently, the precise mechanisms of how these viruses undergo intracellular trafficking to emerge from the infected cell in epithelial tissues is not well understood. Many insights have been made with PRV in animal neurons, both in culture systems and animal models, about the viral genes and host factors involved in these processes. However, understanding of these mechanisms, and the interplay between viral and host proteins, in the human pathogen HSV-1 is even more lacking. Using recombinant fluorescent virus strains of HSV-1 and Total Internal Reflection Microscopy to image the transport of mature viral progeny in epithelial cells, it was determined that the egress of HSV-1 uses constitutive cellular secretory pathways. Specifically, the viral progeny traffic from the trans-Golgi network to the site of exocytosis at the plasma membrane via Rab6a secretory vesicles. This work will contribute to the understanding of how alpha herpesviruses complete their lifecycles in host cells, particularly at the sites where infection initially occurs and can spread to a new organism. Knowledge of these processes may lead to the development of therapeutics or prophylactics to reduce the burden of these viruses.
ContributorsBergeman, Melissa Hope (Author) / Hogue, Ian B (Thesis advisor) / Hogue, Brenda (Committee member) / Roberson, Robert (Committee member) / Varsani, Arvind (Committee member) / Arizona State University (Publisher)
Created2023
Description
The integrin Mac-1 (αMβ2, CD11b/CD18) is an important adhesion receptorexpressed on macrophages and neutrophils. It plays a crucial role in phagocytosis, cell-cell
fusion, and cell migration. αMβ2 is also the most promiscuous integrin with over 100 known
ligands that span a broad range of physical and chemical attributes, many of which bind to
the inserted (I) domain from the αM subunit. The interaction of αMI-domain with cytokine
pleiotrophin (PTN) were determine. PTN is a cationic protein known to induce Mac-1-
mediated adhesion and migration in cells. The data showed that PTN’s interaction with
αMI-domain contains both divalent cation-dependent and independent mechanisms. In
particular, PTN’s N-terminal domain has weak interactions with the N/C-termini side of
αMI-domain using a metal-independent mechanism. However, stronger interaction is
achieved through the chelation of the divalent cation in the metal ion-dependent adhesion
site of active αMI-domain by PTN’s acidic residues. Although many acidic residues in PTN
can act as the chelator, active αMI-domain’s interaction with PTN’s E98 plays an especially
important role. NOE, chemical shift perturbation (CSP) data, and mutagenesis studies
showed residues near E98 are at the binding interface and the E98 mutation greatly reduced
binding affinity between two proteins. Interestingly, the CSP and MD simulation data
showed the binding interface can be supported by the interaction of PTN’s H95 with the
acidic clusters D242, E244, and D273 from αMI-domain, while PTN’s E66 form
electrostatic interaction with R208 and K245 from αMI-domain. The determined
recognition motif of αMI-domain for its ligands is (H/R/K)xxE. The ability to accommodate
the longer distance between E and (H, R, K) compared to the zwitterionic motif RGDii
explained how αMβ2 can interact with a large repertoire of ligands and be versatile in its
functional portfolio.
ContributorsNguyen, Hoa Thi Thanh (Author) / Wang, Xu (Thesis advisor) / Van Horn, Wade (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2024