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- All Subjects: Virology
- Creators: Fromme, Petra
- Resource Type: Text
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
Coronaviruses are a medically significant group of viruses that cause respiratory and enteric infections in humans and a broad range of animals. Coronaviruses assemble at the internal membranes of the endoplasmic reticulum- Golgi intermediate compartment (ERGIC). While there is a basic understanding of how viruses assemble at these membranes, the full mechanistic details are not understood. The coronavirus envelope (E) protein is a small multifunctional viroporin protein that plays a role in virus assembly but its function is unknown. The two goals of this study were : 1. To identify and analyze the localization of MHV E and 2. To identify the functions of conserved residues in the tail of the E protein. This study closely examined the localization, dynamics and mobility of the mouse hepatitis virus (MHV) E protein to gain insight into its functions. The results from the first aim of this study showed that the MHV E protein localizes at the site of assembly in the ERGIC-Golgi region based on analysis by immunofluorescence and correlative electron microscopy. A novel tetra-cysteine tagged MHV E protein was used to study the dynamics of the protein in cells. A recombinant MHV E Lumio virus was used to study the trafficking and mobility of the E protein. Live cell imaging and surface biotinylation confirmed that the E protein does not traffic to the cell surface. Fluorescence recovery after photo-bleaching (FRAP) analyses revealed that the E protein is mobile at the site of localization. As a part of the second aim, conserved prolines and tyrosine in the tail of the protein were targeted by site directed mutagenesis and analyzed for functionality. While none of the residues were absolutely essential for localization or virus production, the mutations had varying degrees of effect on envelope formation, protein stability and virus release. Differential scanning calorimetry data suggests that the proline and tyrosine residues enhance interaction with lipids. A wild type (WT) peptide contained the conserved residues was also able to significantly reduce the hexagonal phase transition temperature of lipids, whereas a mutant peptide with alanine substitutions for the residues did not cause a temperature shift. This suggests that the peptide can induce a negative curvature in lipids. The E protein may be playing a role as a scaffold to allow membrane bending to initiate budding or possibly scission. This data, along with the localization data, suggests that the E protein plays a mechanistic role at the site of virus assembly possibly by remodeling the membrane thereby allowing virus budding and/or scission.
ContributorsVenkatagopalan, Pavithra (Author) / Hogue, Brenda G (Thesis advisor) / Jacobs, Bertram L (Committee member) / Roberson, Robert W. (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2012
Description
Accurate virus detection is important for diagnosis in a timely manner to facilitate rapid interventions and treatments. RNA viruses affect an extensive amount of the world’s population, particularly in tropical countries where emerging infectious agents often arise. Current diagnostic methods have three main problems: they are time consuming, typically not field-portable, and expensive. My research goal is to develop rapid, field-portable and cost sensitive diagnostic methods for RNA viruses. Herein, two different approaches to detect RNA viruses were proposed: Conjugated gold nanoparticles for detection of viral particles or virus-specific antibodies by monitoring changes in their optical properties, and Tentacle Probes coupled with qPCR for detection and differentiation of closely-related viral strains. The first approach was divided into two projects: the study and characterization of the gold nanoparticle-antibody system for detection of virus particles using dynamic light scattering (DLS) and UV-Vis spectrophotometry, and development of a detection method for antibodies using static light scattering (SLS) and antigen-conjugated gold nanoparticles. Bovine serum albumin (BSA) conjugated gold nanoparticles could successfully detect BSA-specific antibodies in vitro, and protein E from Dengue Virus serotype 2 conjugated gold nanoparticles could detect Dengue-specific antibodies, both in vitro and in serum samples. This method is more accurate than currently used detection methods such as dot blots. The second approach uses Tentacle Probes, which are modified molecular beacons, to detect with high specificity two different strains of Lymphocytic Choriomeningitis Virus (LCMV), Armstrong and Clone-13, which differ in only one nucleotide at the target sequence. We successfully designed and use Tentacle Probes for detection of both strains of LCMV, in vitro and in serum from infected mice. Moreover, detection of as little as 10% of Clone-13 strain was possible when diluted in 90% Armstrong strain. This approach enables the detection of different strains of virus even within a mixed quasispecies and may be important for improving intervention strategies for reducing disease. The detection methods provide rapid detection of viruses, including viral strains within mixed populations, and should enhance our ability in providing early responses to emerging infectious diseases due to RNA viruses including Zika or Dengue virus.
ContributorsFranco, Lina Stella (Author) / Mujica, Vladimiro (Thesis advisor) / Blattman, Joseph N (Thesis advisor) / Garcia, Antonio A. (Committee member) / Fromme, Petra (Committee member) / Hayes, Mark (Committee member) / Arizona State University (Publisher)
Created2016
Description
Arachnids belong to the phylum Arthropoda, the largest phylum in the animal kingdom. Ticks are blood-feeding arachnids that vector numerous pathogens of significant medical and veterinary importance, while scorpions have become a common concern in urban desert cities due to the high level of toxicity in their venom. To date, viruses associated with arachnids have been under sampled and understudied. Here viral metagenomics was used to explore the diversity of viruses present in ticks and scorpions. American dog ticks (Dermacentor variabilis) and blacklegged ticks (Ixodes scapularis) were collected in Pennsylvania while one hairy scorpion (Hadrurus arizonensis) and four bark scorpions (Centruroides sculpturatus) were collected in Phoenix. Novel viral genomes described here belong to the families Polyomaviridae, Anelloviridae, Genomoviridae, and a newly proposed family, Arthropolviridae.
Polyomaviruses are non-enveloped viruses with a small, circular double-stranded DNA (dsDNA) genomes that have been identified in a variety of mammals, birds and fish and are known to cause various diseases. Arthropolviridae is a proposed family of circular, large tumor antigen encoding dsDNA viruses that have a unidirectional genome organization. Genomoviruses and anelloviruses are ssDNA viruses that have circular genomes ranging in size from 2–2.4 kb and 2.1–3.8 kb, respectively. Genomoviruses are ubiquitous in the environment, having been identified in a wide range of animal, plant and environmental samples, while anelloviruses have been associated with a plethora of animals.
Here, 16 novel viruses are reported that span four viral families. Eight novel polyomaviruses were recovered from bark scorpions, three arthropolviruses were recovered from dog ticks and one arthropolvirus from a hairy scorpion. Viruses belonging to the families Polyomaviridae and Arthropolviridae are highly divergent. This is the first more extensive study of these viruses in arachnids. Three genomoviruses were recovered from both dog and deer ticks and one anellovirus was recovered from deer ticks, which are the first records of these viruses being recovered from ticks. This work highlights the diversity of dsDNA and ssDNA viruses in the arachnid population and emphasizes the importance of performing viral surveys on these populations.
Polyomaviruses are non-enveloped viruses with a small, circular double-stranded DNA (dsDNA) genomes that have been identified in a variety of mammals, birds and fish and are known to cause various diseases. Arthropolviridae is a proposed family of circular, large tumor antigen encoding dsDNA viruses that have a unidirectional genome organization. Genomoviruses and anelloviruses are ssDNA viruses that have circular genomes ranging in size from 2–2.4 kb and 2.1–3.8 kb, respectively. Genomoviruses are ubiquitous in the environment, having been identified in a wide range of animal, plant and environmental samples, while anelloviruses have been associated with a plethora of animals.
Here, 16 novel viruses are reported that span four viral families. Eight novel polyomaviruses were recovered from bark scorpions, three arthropolviruses were recovered from dog ticks and one arthropolvirus from a hairy scorpion. Viruses belonging to the families Polyomaviridae and Arthropolviridae are highly divergent. This is the first more extensive study of these viruses in arachnids. Three genomoviruses were recovered from both dog and deer ticks and one anellovirus was recovered from deer ticks, which are the first records of these viruses being recovered from ticks. This work highlights the diversity of dsDNA and ssDNA viruses in the arachnid population and emphasizes the importance of performing viral surveys on these populations.
ContributorsSchmidlin, Kara (Author) / Varsani, Arvind (Thesis advisor) / Van Doorslaer, Koenraad (Committee member) / Stenglein, Mark (Committee member) / Arizona State University (Publisher)
Created2019
Description
This work comprises a cumulative effort to provide analysis of proteins relevant to understanding and treating human disease. This dissertation focuses on two main protein complexes: the structure of the Chimp adenovirus Y25 capsid assembly, as used in the SARS-CoV-2 vaccine, Vaxzveria, and the Dbl family RhoGEF (guanosine exchange factor) Syx and its associated small G protein, RhoA. The course of research was influenced heavily by the onset of the Covid-19 pandemic and associated lockdown, which pushed anyone with the means to do meaningful research to shift priorities towards addressing the greatest public health crisis since the 1918 flu pandemic.
Analysis of the Syx-RhoA complex for the purposes of structurally guided drug design was initially the focus of heavy optimization efforts to overcome the numerous challenges associated with expression, purification, and handling of this protein. By analyzing E. Coli derived protein new important knowledge was gained about this protein’s biophysical characteristics which contribute to its behavior and may inform drug design efforts. Expression in SF9 insect cells resulted in promising conditions for production of homogeneous and monodispersed protein. Homology modeling and molecular dynamics simulation of this protein support hypotheses about its interactions with both RhoA as well as regions of the cytoplasmic leaflet of the cell membrane.
Structural characterization of ChAdOx1, the adenoviral vector used in the AstraZeneca Covid-19 vaccine, Vaxzveria resulted in the highest resolution adenovirus structure ever solved (3.07Å). Subsequent biochemical analysis and computational simulations of PF4 with the ChAdOx1 capsid reveal interactions with important implications for vaccine induced thrombocytic throbocytopenia syndrome, a disorder observed in approximately 0.000024% of patients who receive Vaxzveria.
ContributorsBoyd, Ryan J (Author) / Fromme, Petra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2021