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Title: The use of synthetic biology to engineer vaccines against Ebola and Zika viruses using Salmonella based delivery systems
Author: Kildani, Rebecca Sarah
ISNI:       0000 0004 8505 8729
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2019
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Ebola and Zika viruses have been brought to the world's attention in recent years due to widespread outbreaks and the devastating diseases they cause in humans. Hence there is an urgent need to develop vaccines which can protect from disease. Salmonella vaccine strains have been genetically attenuated so as not to cause disease. These vaccine strains can be engineered to deliver heterologous antigens from viruses, bacteria, and worms to the host's immune system to elicit protective immunity. Such vaccines can be orally administered and would not need cold storage. A successful vaccine using this platform would have the additional advantages of being trivalent, protecting against Salmonella, Tetanus and Ebola or Zika virus. The Ebola Glycoprotein (GP) and the Zika envelope protein (ZE) contain important protective epitopes, which are targets for neutralising antibodies, and therefore considered prime targets for vaccine development. Using the Salmonella based delivery systems we have successfully expressed these Ebola and Zika virus vaccine candidate proteins for delivery to the immune system. A large panel of 19 variant constructs has been generated, which express these viral antigens, or sub-fragments containing important protective epitopes, as fusions to the Cfragment of Tetanus Toxin (TetC). TetC is a potently immunogenic molecule, which can be expressed to high levels by Salmonella and helps to solubilise the expressed fusion protein partner and act as an adjuvant. Expression of proteins in this vector is driven by the in vivo inducible promoter, nirB. The panel of GP and ZE genes and cassettes were synthesised to be codon optimised, with toxic hydrophobic signal sequence and transmembrane regions removed, for expression in Salmonella Typhimurium. By testing with western blot, it was found that TetC rescued expression of the GP and ZE proteins, which were either expressed at much lower levels or not at all in constructs lacking the TetC fusion partner. The most promising candidates were then taken forward to in vivo testing. These strains have been shown to be stable both in vitro and in vivo, retaining the expression vector, and still expressing the antigens after passage in BALB/c mice. Vaccination experiments were then carried out in BALB/c mice and have unfortunately shown that after a single immunisation, there was no significant IgG antibody response to the GP or ZE antigens. An alternative non-living Salmonella vaccine delivery platform has also been investigated. By making tolR and mlaA gene knockouts in Salmonella Typhimurium strain SL1344, these strains have been shown to produce elevated amounts of outer membrane vesicles iii (OMVs) than their wild type counterparts. These immunogenic vesicles could then be used to carry antigen as cargo for vaccine delivery and subsequently purified for immunisation. Constructs were made to target the expression of the Zika envelope protein to the outer membrane or periplasm, using signal sequences from OmpA and DsbA respectively. The knockout strain SL1344 ΔtolR, harbouring these plasmid constructs, was able to express ZE in OMVs, hopefully either decorating the OMV surface, or contained within the lumen. It is hoped that this could lead to an improved immune response compared to the live platform, as an adequate antigenic dose could be pre-determined and administered accordingly. The outer membrane components (such as LPS) which are naturally included in the OMVs, act as an adjuvant, and thus can increase the immunogenicity and efficacy of such a platform. A wider range of potential recipients could also be reached with such a vaccine, as vulnerable populations such as the very old, very young or immunocompromised patients cannot receive live vaccines. This study has shown the feasibility of such an approach of targeting antigen cargo to the OMVs, and will hopefully pave the way for future exciting research to determine its effectiveness.
Supervisor: Not available Sponsor: Barbour Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available