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- All Subjects: Vaccines
- Creators: Hogue, Brenda
- Status: Published
Biological and immunological characterization of plant-produced HIV-1 Gag/dgp41 virus-like particles
In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression.
Current methods for IgG antibody detection include enzyme immunoassays (EIA) such as the commercially available Diamedix Immunosimplicity® Measles IgG test kit and the Diamedix Immunosimplicity® Mumps IgG test kit. EIAs generally provide high sensitivity and strong specificity, however, there is a need for rapid screening of measles and mumps specific immunity in outbreak and resource-limited areas which could be solved by use a point-of-care (POC) platform.
This study aims to optimize a point-of-care device for the multiplexed detection of MeV, MuV, and RuV IgG antibodies in sera and to compare the sensitivity to commercial enzyme immunoassays. The IgG antibody levels to MeV and MuV were measured using EIA test kits for a total of 44 healthy serum samples. Of the samples, 6% were seronegative for MeV-specific IgG antibodies and 75% were seronegative for MuV-specific antibodies, showing low correlation of IgG antibody levels between both viruses.
To improve the sensitivity of the POC device, multiple conjugated fluorescent secondary antibodies were tested with different surface chemistries. Signal detection was measured using the pre-developed four-site slide reader. Preliminary data show that Nile Red microspheres provide robust signal detection and should be the secondary antibody of choice when sera are tested for IgG antibodies using the POC platform in future work.
DNA nanotechnology is ideally suited for numerous applications from the crystallization and solution of macromolecular structures to the targeted delivery of therapeutic molecules. The foundational goal of structural DNA nanotechnology was the development of a lattice to host proteins for crystal structure solution. To further progress towards this goal, 36 unique four-armed DNA junctions were designed and crystallized for eventual solution of their 3D structures. While most of these junctions produced macroscale crystals which diffracted successfully, several prevented crystallization. Previous results used a fixed isomer and subsequent investigations adopted an alternate isomer to investigate the impact of these small sequence changes on the stability and structural properties of these crystals. DNA nanotechnology has also shown promise for a variety biomedical applications. In particular, DNA origami has been demonstrated as a promising tool for targeted and efficient delivery of drugs and vaccines due to their programmability and addressability to suit a variety of therapeutic cargo and biological functions. To this end, a previously designed DNA barrel nanostructure with a unique multimerizable pegboard architecture has been constructed and characterized via TEM for later evaluation of its stability under biological conditions for use in the targeted delivery of cargo, including CRISPR-containing adeno-associated viruses (AAVs) and mRNA.