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- All Subjects: Influenza
- Creators: Legutki, Bart
- Creators: Mor, Tsafrir
Description
Influenza is a deadly disease for which effective vaccines are sorely lacking. This is largely due to the phenomena of antigenic shift and drift in the influenza virus's surface proteins, hemagglutinin (HA) and neuraminidase (NA). The ectodomain of the matrix 2 protein (M2e) of influenza A, however, has demonstrated high levels of conservation. On its own it is poorly immunogenic and offers little protection against influenza infections, but by combining it with a potent adjuvant, this limitation may be overcome. Recombinant immune complexes, or antigens fused to antibodies that have been engineered to form incredibly immunogenic complexes with one another, were previously shown to be useful, immunogenic platforms for the presentation of various antigens and could provide the boost in immunogenicity that M2e needs to become a powerful universal influenza A vaccine. In this thesis, genetic constructs containing geminiviral replication proteins and coding for a consensus sequence of dimeric M2e fused to antibodies featuring complimentary epitopes and epitope tags were generated and used to transform Agrobacterium tumefaciens. The transformed bacteria was then used to cause Nicotiana benthamiana to transiently express M2e-RICs at very high levels, with enough RICs being gathered to evaluate their potency in future mouse trials. Future directions and areas for further research are discussed.
ContributorsFavre, Brandon Chetan (Author) / Mason, Hugh (Thesis director) / Mor, Tsafrir (Committee member) / Diamos, Andrew (Committee member) / Department of Psychology (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The influenza virus is the main cause of thousands of deaths each year in the United States, and far more hospitalizations. Immunization has helped in protecting people from this virus and there are a number of therapeutics which have proven effective in aiding people infected with the virus. However, these therapeutics are subject to various limitations including increased resistance, limited supply, and significant side effects. A new therapeutic is needed which addresses these problems and protects people from the influenza virus. Synbodies, synthetic antibodies, may provide a means to achieve this goal. Our group has produced a synbody, the 5-5 synbody, which has been shown to bind to and inhibit the influenza virus. The direct pull down and western blot techniques were utilized to investigate how the synbody bound to the influenza virus. Our research showed that the 5-5 synbody bound to the influenza nucleoprotein (NP) with a KD of 102.9 ± 74.48 nM. It also showed that the synbody bound strongly to influenza viral extract from two different strains of the virus, the Puerto Rico (H1N1) and Sydney (H3N2) strains. This research demonstrated that the 5-5 synbody binds with high affinity to NP, which is important because influenza NP is highly conserved between various strains of the virus and plays an important role in the replication of the viral genome. It also demonstrated that this binding is conserved between various strains of the virus, indicating that the 5-5 synbody potentially could bind many different influenza strains. This synbody may have potential as a therapeutic in the future if it is able to demonstrate similar binding in vivo.
ContributorsKombe, Albert E. (Author) / Diehnelt, Chris (Thesis director) / Woodbury, Neal (Committee member) / Legutki, Bart (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of International Letters and Cultures (Contributor)
Created2014-05
Description
The influenza virus, also known as "the flu", is an infectious disease that has constantly affected the health of humanity. There is currently no known cure for Influenza. The Center for Innovations in Medicine at the Biodesign Institute located on campus at Arizona State University has been developing synbodies as a possible Influenza therapeutic. Specifically, at CIM, we have attempted to design these initial synbodies to target the entire Influenza virus and preliminary data leads us to believe that these synbodies target Nucleoprotein (NP). Given that the synbody targets NP, the penetration of cells via synbody should also occur. Then by Western Blot analysis we evaluated for the diminution of NP level in treated cells versus untreated cells. The focus of my honors thesis is to explore how synthetic antibodies can potentially inhibit replication of the Influenza (H1N1) A/Puerto Rico/8/34 strain so that a therapeutic can be developed. A high affinity synbody for Influenza can be utilized to test for inhibition of Influenza as shown by preliminary data. The 5-5-3819 synthetic antibody's internalization in live cells was visualized with Madin-Darby Kidney Cells under a Confocal Microscope. Then by Western Blot analysis we evaluated for the diminution of NP level in treated cells versus untreated cells. Expression of NP over 8 hours time was analyzed via Western Blot Analysis, which showed NP accumulation was retarded in synbody treated cells. The data obtained from my honors thesis and preliminary data provided suggest that the synthetic antibody penetrates live cells and targets NP. The results of my thesis presents valuable information that can be utilized by other researchers so that future experiments can be performed, eventually leading to the creation of a more effective therapeutic for influenza.
ContributorsHayden, Joel James (Author) / Diehnelt, Chris (Thesis director) / Johnston, Stephen (Committee member) / Legutki, Bart (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
Influenza is a deadly disease that poses a major threat to global health. The surface proteins of influenza A, the type most often associated with epidemics and pandemics, mutate at a very high frequency from season to season, reducing the efficacy of seasonal influenza vaccines. However, certain regions of these proteins are conserved between strains of influenza A, making them attractive targets for the development of a ‘universal’ influenza vaccine. One of these highly conserved regions is the ectodomain of the influenza matrix 2 protein (M2e). Studies have shown that M2e is poorly immunogenic on its own, but when properly adjuvanted it can be used to induce protective immune responses against many strains of influenza A. In this thesis, M2e was fused to a pair experimental ‘vaccine platforms’: an antibody fusion protein designed to assemble into a recombinant immune complex (RIC) and the hepatitis B core antigen (HBc) that can assemble into virus-like particles (VLP). The two antigens were produced in Nicotiana benthamiana plants through the use of geminiviral vectors and were subsequently evaluated in mouse trials. Mice were administered three doses of either the VLP alone or a 1:1 combination of the VLP and the RIC, and recipients of both the VLP and RIC exhibited endpoint anti-M2e antibody titers that were 2 to 3 times higher than mice that received the VLP alone. While IgG2a:IgG1 ratios, which can suggest the type of immune response (TH1 vs TH2) an antigen will elicit, were higher in mice vaccinated solely with the VLP, the higher overall titers are encouraging and demonstrate a degree of interaction between the RIC and VLP vaccines. Further research is necessary to determine the optimal balance of VLP and RIC to maximize IgG2a:IGg1 ratios as well as whether such interaction would be observed through the use of a variety of diverse antigens, though the results of other studies conducted in this lab suggests that this is indeed the case. The results of this study demonstrate not only the successful development of a promising new universal influenza A vaccine, but also that co-delivering different types of recombinant vaccines could reduce the total number of vaccine doses needed to achieve a protective immune response.
ContributorsFavre, Brandon Chetan (Author) / Mason, Hugh S (Thesis advisor) / Mor, Tsafrir (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2019