The influenza A virus is responsible for 250,000 to 500,000 deaths every year worldwide and millions more could die in the event of a serious pandemic. Vaccines against influenza have existed for long, but until today they have been limited by extensive production times and reduced cross-protection between different strains of the virus. This leads to a recurrent need to update the vaccine composition every year, which is both costly and inadequate to fight pandemics. An innovative approach that could improve the vaccine efficacy has been recently developed based on the selection of conserved influenza epitopes with potential to induce broader immune responses. The 23-amino acid extracellular domain of the M2 protein (M2e) is highly conserved among different influenza A strains and thus it seems like an ideal candidate for a universal influenza vaccine. However, due to its small size, it is a poor immunogen when used on its own. The aim of this project was to produce M2e-presenting virus-like particles (VLPs) in Nicotiana benthamiana plants via Agrobacterium-mediated transient expression. Plants are increasingly being examined as alternative recombinant protein expression systems due to their safety, scalability and rapid production times. Moreover, numerous studies suggest the use of recombinant virus-like particles (VLPs) to increase the immunogenicity of antigens. Therefore, to obtain VLPs presenting the M2e epitope, I genetically engineered several different M2e-HA fusion proteins by replacing the hemagglutinin (HA) globular head and main epitope with five tandem repeats of M2e epitope sequences (5xM2e) from human, swine, and avian origin influenza A viruses. To increase the chances of obtaining VLPs, M2e-HA fusions either contained the HA stalk domain (5xM2e-HAstalk) or simply the transmembrane region (5xM2e-HAtrans). Furthermore, the tetramerizing leucine zipper derived from the General Control Protein (GCN4) was also included in some of the constructs to promote particle formation. In total, six different M2e-HA fusions were created: 5xM2e-GCN4-HAstalk, 5xM2e-GCN4-HAtrans, 5xM2e-HAstalk, 5xM2e-HAtrans, 1xM2e-HAstalk and 1xM2e-HAtrans. The expression of these proteins was optimized in plants by testing different conditions and using three different expression vectors. Overall, I was able to show expression after only 3 days post-infiltration for most of the M2e-HA v fusion proteins utilizing the pEAQ-HT and pRIC 3.0 expression vectors whereas expression levels with pTRAc were low or non-detectable. Once the expression of the M2e-HA fusions was optimized, the two proteins with the highest potential to form VLPs were selected for further characterization (5xM2e-HAstalk and 5xM2eHAtrans). Using transmission electron microscopy to analyse purified proteins, both 5xM2eHAstalk and 5xM2e-HAtrans were shown to assemble into VLPs resembling the shape and size of native HA VLPs. These VLPs could also be observed in the apoplastic fractions of infiltrated leaves. However, due to the low number of particles observed, the successful incorporation of the M2e peptide on the surface of the particles was inconclusive, as shown by M2e-specific immuno-gold labelling experiments. Furthermore, contrarily to previous studies, co-expression of the M2e-HA fusions with the M1 protein resulted in a decrease in recombinant protein accumulation and VLP formation in our plant system. A possible inhibition mechanism by the M1 protein is discussed. In summary, this research provides preliminary data to produce universal influenza vaccines in plants. I report here for the first time that M2e fused to either the stalk or transmembrane domain of the HA protein, can self-assemble into VLPs without any other proteins, in N. benthamiana plants. Future work on the immunogenicity of the VLPs produced in this study is required to confirm their potential as a universal influenza vaccine that can be rapidly produced.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/27063 |
| Date | January 2017 |
| Creators | De Figueiredo Pinto Gomes Pera, Francisco |
| Contributors | Hitzeroth, Inga I, Rybicki, Edward P |
| Publisher | University of Cape Town, Faculty of Science, Department of Molecular and Cell Biology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Master Thesis, Masters, MSc |
| Format | application/pdf |
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