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Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E) protein as an immunogen to be vectored by a measles virus (MV) vaccine. However the domain III (DIII) of the

Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E) protein as an immunogen to be vectored by a measles virus (MV) vaccine. However the domain III (DIII) of the dengue 2 E protein is too small to be immunogenic by itself. In order for it to be displayed on a larger particle, it was inserted into the amino terminus of small hepatitis B surface antigen (HBsAg, S) coding sequence. To generate the recombinant MV vector and verify the efficiency of this concept, a reverse genetics system was used where the MV vectors express one or two additional transcription units to direct the assembly of hybrid HBsAg particles. Two types of recombinant measles virus were produced: pB(+)MVvac2(DIII-S,S)P and pB(+)MVvac2(DIII-S)N. Virus recovered from pB(+)MVvac2(DIII-S,S)P was viable. An ELISA assay was performed to demonstrate the expression and secretion of HBsAg. Supernatant from MVvac2(DIII-S,S)P infected cells confirmed that hybrid HBsAg-domain III particles with a density similar to traditional HBsAg particles were released. Characteristics of the subviral particle have been analyzed for the successful incorporation of domain III. The replication fitness of the recombinant MV was evaluated using multi-step growth kinetics and showed reduced replication fitness when compared to the parental strain MVvac2. This demonstrates that viral replication is hindered by the addition of the two inserts into MV genome. Further analysis of MVvac2(DIII-S)N is needed to justify immune response studies in a small animal model using both of the generated recombinant vectors.
ContributorsHarahap, Indira Saridewi (Author) / Reyes del Valle, Jorge (Thesis director) / Hogue, Brenda (Committee member) / Misra, Rajeev (Committee member) / Barrett, The Honors College (Contributor) / T. Denny Sanford School of Social and Family Dynamics (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor)
Created2014-05
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Plant-made virus-like particles (VLPs), composed of HIV-1 Gag and deconstructed gp41 proteins, have been shown to be safe and immunogenic in mice. Here, we report the successful production of HIV-1 Gag/dgp41 VLPs in Nicotiana benthamiana, using an enhanced geminivirus-based expression vector. This novel vector results in unique expression kinetics, with

Plant-made virus-like particles (VLPs), composed of HIV-1 Gag and deconstructed gp41 proteins, have been shown to be safe and immunogenic in mice. Here, we report the successful production of HIV-1 Gag/dgp41 VLPs in Nicotiana benthamiana, using an enhanced geminivirus-based expression vector. This novel vector results in unique expression kinetics, with peak protein accumulation and minimal necrosis achieved on day 4 post-infiltration. In comparing various purification strategies, it was determined that a 20% ammonium sulfate precipitation is an effective and efficient method for removing plant proteins and purifying the recombinant VLPs of interest. If further purification is required, this may be achieved through ultracentrifugation. VLPs are a useful platform for a variety of biomedical applications and developing the technology to efficiently produce VLPs in the plant expression system is of critical importance.

ContributorsFleming, Claire (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Kamzina, Aigerim (Committee member) / Barrett, The Honors College (Contributor) / Department of Physics (Contributor) / School of Life Sciences (Contributor)
Created2022-05
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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

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