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- Creators: Arizona State University
- Creators: Stabenfeldt, Sarah
- Status: Published
Description
Nearly four decades after HIV (Human Immunodeficiency Virus) was identified as the causal agent of the AIDS (Acquired Immunodeficiency Syndrome) pandemic, it remains a top global health concern impacting millions of people around the world particularly in Sub-Saharan Africa. Despite significant scientific, governmental and nongovernmental organizational efforts, most HIV-infected patients do not have access to prevention and treatment. Since cure is not available yet, developing a vaccine to prevent HIV from spreading is a priority. Previous studies have worked on an HIV vaccine platform using attenuated Vaccinia vector and plant-produced HIV virus-like particles (VLPs) to deliver Gag and dgp41 antigens as a heterologous prime-boost strategy. To further study this work, I conducted immunogenicity studies in rabbits which exhibited high IgG responses against Gag (p < 0.002) and less to dgp41. To increase the immunogenicity to dgp41, focusing on MPER, a combination of IgG fusions with VLPs as a vaccine platform was studied in mice. Both IgG fusion constructs showed similar serum results, though Gag-specific serum IgG responses were significantly higher (p < 0.007) for the recombinant immune complex (RIC) group than hexamer forming complexes (Hex). In an effort to expand the use of HIV VLPs, RSV (Respiratory Syncytial Virus) pre-fusion stabilized F (pre-F) protein was presented by self-assembling HIV-1 Gag as a potential vaccine strategy for RSV infections. Multiple constructs were designed to assemble into chimeric VLPs and tested for recombinant plant expression. Mouse immunogenicity study using these chimeric VLPs showed significantly high F-specific IgG (p < 0.001) in serum and superior IgA in mucosal samples for the group that received one of the pre-F stabilized VLP constructs. Moreover, when the same antigen was administered with cholera toxin intranasally, it generated IgA response in nasal flush higher than when it was administered subcutaneously. To summarize, this study showed the efficiency of a plant-produced VLP-based system as an adaptable chimeric vaccine platform for potential use with various viral antigens in pursuit of a vaccine strategy that is immunogenic in animal studies.
ContributorsKamzina, Aigerim (Author) / Mor, Tsafrir TM (Thesis advisor) / Mason, Hugh HM (Committee member) / Jacobs, Bertram BJ (Committee member) / Blattman, Joseph JB (Committee member) / Arizona State University (Publisher)
Created2022
Description
Recent studies in traumatic brain injury (TBI) have found a temporal window where therapeutics on the nanometer scale can cross the blood-brain barrier and enter the parenchyma. Developing protein-based therapeutics is attractive for a number of reasons, yet, the production pipeline for high yield and consistent bioactive recombinant proteins remains a major obstacle. Previous studies for recombinant protein production has utilized gram-negative hosts such as Escherichia coli (E. coli) due to its well-established genetics and fast growth for recombinant protein production. However, using gram-negative hosts require lysis that calls for additional optimization and also introduces endotoxins and proteases that contribute to protein degradation. This project directly addressed this issue and evaluated the potential to use a gram-positive host such as Brevibacillus choshinensis (Brevi) which does not require lysis as the proteins are expressed directly into the supernatant. This host was utilized to produce variants of Stock 11 (S11) protein as a proof-of-concept towards this methodology. Variants of S11 were synthesized using different restriction enzymes which will alter the location of protein tags that may affect production or purification. Factors such as incubation time, incubation temperature, and media were optimized for each variant of S11 using a robust design of experiments. All variants of S11 were grown using optimized parameters prior to purification via affinity chromatography. Results showed the efficiency of using Brevi as a potential host for domain antibody production in the Stabenfeldt lab. Future aims will focus on troubleshooting the purification process to optimize the protein production pipeline.
ContributorsEmbrador, Glenna Bea Rebano (Author) / Stabenfeldt, Sarah (Thesis director) / Plaisier, Christopher (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05