Generation of influenza A virus vectors for the delivery of antigenic proteins

Karkashan, A 2016, Generation of influenza A virus vectors for the delivery of antigenic proteins, Doctor of Philosophy (PhD), Applied Sciences, RMIT University.

Document type: Thesis
Collection: Theses

Attached Files
Name Description MIMEType Size Video - HA wild-type protein MD simulation - side view Click to show the corresponding preview/stream video/quicktime 4.23MB Video - HA wild-type protein MD simulationm - top view Click to show the corresponding preview/stream video/quicktime 4.46MB Video - HA-His protein MD simulation - side view Click to show the corresponding preview/stream video/quicktime 7.34MB Video - HA-His protein MD simulation - top view Click to show the corresponding preview/stream video/quicktime 5.61MB
Karkashan.pdf Thesis application/pdf 11.82MB
Title Generation of influenza A virus vectors for the delivery of antigenic proteins
Author(s) Karkashan, A
Year 2016
Abstract Viruses are highly efficient at infecting cells. They overcome the cellular barrier and transfer their genetic material into the host cell nucleus, and efficiently use the cellular machinery for the replication and production of new virions (Gale et al, 2000; Brave et al, 2006). Viruses have been widely studied as naturally existing vehicles, and for utilization for the delivery of heterologous antigens to the immune system (Souza et al, 2005; Brave et al, 2006; Ura et al, 2014).

The development of reverse genetics for negative-strand RNA viruses made it possible to manipulate the influenza virus genome (Stukova et al, 2006). The influenza virus offers several advantages as an antigen delivery vector, including safety profiles and the virus’s ability to induce strong cellular and humoral immune responses (Stukova et al, 2006; Martinez-Sobrido & Garcia-Sastre, 2007).

There are two main reverse genetics systems that can be used for recombinant influenza virus generation, and for this study the helper virus-based method was used. Development of an effective selection system to isolate the generated recombinant influenza virus from the background of helper influenza virus is essential as the first step to continue the development of influenza viral vector vaccines. The widely used isolation technique utilising insertion of affinity tags within the desired protein is one very powerful method (Bornhorst & Falke, 2000). Theoretically, inserting an affinity tag within a selected influenza protein could be an applicable approach for the purification of the generated recombinant virus.

This study investigates the possibility of generating His tagged influenza virus, which can be positively selected using the Immobilized-metal affinity chromatography (IMAC) technique. The successful production of tagged influenza virus will help to overcome the issue of selection for the desired recombinant virus (i.e., expressing immunogenic antigen) using the helper virus-based system.

The first objective of this study was to design a gene structure for tagged influenza hemagglutinin (HA) gene. The His tag sequence was placed within the HA1 subunit, specifically within the Ca2 antigenic region gene between amino acid 158 and 159. The HA gene used for this study was from H1N1 influenza virus. Mapping of the HA sequence features suggested that the selected insertion site was unlikely to interfere with any vital regions. Also, building a structural model for the recombinant HA predicted that the His tag was placed within an exposed HA domain. Finally, the HA-His6 pol I construct was successfully designed, and synthesis and cloning of the recombinant HA gene-construct into DNA plasmid was completed.

The second objective of this study was to generate a recombinant influenza virus using helper virus-based reverse genetics. Influenza virus (A/Puerto Rico/8/34, H1N1) propagation was achieved using MDCK cells, and a reasonably high virus titer was obtained. MDCK cells were also used for the generation of recombinant influenza virus. Around 40% of the cells were successfully transfected with the plasmid containing the HA-His6 pol I construct (canine RNA polymerase I), which was assessed by using EGFP plasmid as an indicator of transfection efficiency. Co-infecting the cells with the helper virus (wild-type PR8) at MOI 0.1 resulted in the generation of His tagged influenza virus. The presence of the recombinant virus was confirmed using the PCR detection technique, which was performed afterward when immunodetection by anti-His antibody failed to detect the His tagged protein.

The third objective of this study was to further investigate the availability and stability of the His tagged influenza virus. As the first anti-His antibody failed to recognise the generated His tagged virus, another anti-His with high affinity and specificity was used. However, immunorecognition failed using this other antibody as well. Moreover, using the highly sensitive enhanced chemiluminescent assays (ECL) assay for developing the Western blot membrane also resulted in unsuccessful detection. A recent tertiary model structure (2014) showed a different His tag folding than what was predicted in the earliest model (2011) within the HA protein. The latest model prediction resulted in a partially hidden His epitope, and this was in agreement with the solvent accessibility value predicted by the I-TASSER server. After performing molecular dynamics simulation, the results obtained suggested that there was no significant difference between the recombinant HA and the wild-type HA proteins in terms of stability. Availability of the recombinant virus was confirmed after passaging the virus mixture (recombinant and wild-type viruses) for up to three rounds. The final investigation in this study was performed to enhance the production of the recombinant virus amongst the wild-type one. Again, this trial did not improve on antibody detection.

Taken together, these results indicate that recombinant influenza virus incorporating a His tag could be generated using the helper virus-based system. Also, the recombinant virus can replicate among the wild-type viruses, and it can be stable for several passages. As there were many factors that might lead to unsuccessful immunodetection, it is still possible to generate a tagged influenza virus that can be successfully detected by antibody, because there are other alternatives to investigate in the future. Studying the HA sequence features and the possible sites to insert the affinity tag, as well as the structural prediction in this study, provided useful information for the future incorporation and expression of pathogenic or disease proteins within the HA protein. The investigations that were undertaken in this study have resulted in new knowledge in the field of recombinant influenza virus.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Applied Sciences
Subjects Environmental Molecular Engineering of Nucleic Acids and Proteins
Keyword(s) Recombinant influenza virus
Reverse genetics
His tag
Molecular dynamics simulation
Helper virus-based reverse genetics
Genetic engineering
Viral vectors vaccines
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Created: Wed, 24 Aug 2016, 15:06:07 EST by Keely Chapman
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