The development of a safe and effective HIV vaccine remains challenging due to its high antigenic variability. Poxviruses are large, stable, and have a track record of use as human vaccine candidates. Recombinant fowlpox virus 9 (rFP9), a highly attenuated host range-restricted poxvirus strain, has been safely administered to humans with no ill effects, and is known to be immunogenic. This thesis describes the construction of complex rFP9 encoding various sequences of SIVmac239. The SIVmac239/macaque model is widely used for HIV vaccine development. The ultimate aim of this work was to combine the advantages of FP9 with those of live attenuated SIV to produce a safe yet hopefully effective model HIV vaccine candidate. Transfer plasmids for five different insertion sites within the FP9 genome were designed and constructed. Homologous recombination (HR) of adjacent FP9 sequences was employed to facilitate the integration of SIVmac239 sequences into the FP9 genome. Positive rFP9 were identified by blue colouration in presence of X-gal using a transient colour selection (TCS) technique, and the final markerless pure recombinants were confirmed by PCR. Expression of the target SIV proteins in the presence of T7 polymerase has been demonstrated by immunocytochemical (ICC) staining and Western blotting (WB) assays. Expression was also quantified by enzyme-linked immunosorbent assay (ELISA) in various cell lines at multiple time points. Five different unique rFP9 have been constructed through this project. All SIVmac239 open reading frames (ORFs) save nef have been integrated into the FP9 genome, and protein expression demonstrated where possible. Moreover, a single rFP9 vector expressing the defective SIVmac239 genome driven by T7 RNA polymerase has been successfully constructed and validated using a green fluorescent protein marker.rFP9 showed appropriate transgene expression in both avian and mammalian cells, although at different levels. The expression efficiency of rFP9 was finally compared to another attenuated poxvirus vector, modified vaccinia Ankara (MVA). Comparing the protein expression levels between rFP9 and rMVA was quite difficult because different poxvirus promoters (early/late in rFP9; intermediate in rMVA) were used to direct the transcription of the T7 RNA gene. Given this limitation, although generally higher levels of expression were seen with rFP9, this cannot be attributed to the FP9 with any certainty.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:689575 |
| Date | January 2016 |
| Creators | Alsafi, Radi Taha M. |
| Contributors | Vallely, Pamela ; Blanchard, Thomas ; Cholapura-Puttaswama, Cpc Gowda |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/generation-of-complex-recombinant-fowlpox-virus-9-fp9-encoding-simian-immunodeficiency-virus-sivmac239-sequences-as-a-model-hiv-vaccine-candidate(1a015762-8dc2-4153-a586-d7fab88b9658).html |
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