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dc.contributor.advisorBurt, Felicity Jane
dc.contributor.authorViljoen, Natalie
dc.date.accessioned2016-01-11T09:23:27Z
dc.date.available2016-01-11T09:23:27Z
dc.date.issued2014-12
dc.identifier.urihttp://hdl.handle.net/11660/2103
dc.description.abstractEnglish: Yellow fever virus (YFV), a mosquito-borne virus that belongs to the family Flaviviridae and genus Flavivirus, is a significant cause of morbidity and mortality in yellow fever endemic areas, especially in West Africa. In humans, YFV causes yellow fever, a disease characterised by renal failure, jaundice, and/or haemorrhage. The burden of disease is highest in Africa constituting approximately 90% of reported cases worldwide. Despite the availability of highly efficacious live attenuated vaccines against YFV, the estimated prevalence for yellow fever in Africa was 130 000 severe cases and 78 000 deaths for 2013. The available live attenuated vaccines have been contraindicated for use in immunocompromised patients and individuals with hypersensitivity to eggs and/or chicken. Vaccine-associated neurotropic adverse events that result in the development of meningoencephalitis in infants and vaccine-associated viscerotropic adverse events that result in disease resembling wild-type yellow fever have been reported. Vaccine-associated viscerotropic adverse events are associated with fatality rates exceeding 40%. Therefore, there is a need for a safer alternative to complement the use of the available live attenuated vaccines. The aim of this study was to construct a DNA-launched candidate vaccine against YFV and to determine the immunogenicity of the DNA-launched pSinED-lll replicon, which would provide information regarding the applicability of DNA-launched replicons as an approach to vaccine development. The pSinGFP replicon encoding the green fluorescent protein (GFP) was kindly provided by Prof. Mark Heise. Expression of the GFP was confirmed in mammalian cell culture post-transfection with the pSinGFP replicon, thus confirming the functioning of the replicon elements and subsequent expression of the encoded protein. The gene encoding the GFP was excised and replaced with a synthesised codon-optimised gene encoding the YFV ED-lll protein using directional cloning. The nucleotide sequence was confirmed by bidirectional sequencing at the site of insertion and subsequently protein expression was confirmed in selected mammalian cell lines. Protein expression was confirmed by detection of the C-terminal histidine tag by immunofluorescence using anti-His6 mouse monoclonal antibody. Thereafter, the expressed protein was characterised by immunofluorescence using mouse immune sera previously shown to contain anti-YFV ED-lll antibody. Reactivity of the expressed protein with anti-YFV ED-lll antibody substantiated the use of the pSinED-lll replicon in a mouse immunisation study. Mice were immunised intramuscularly according to pre-approved immunisation regimes consisting of DNA only, as well as mixed modal immunisation regimes. Serum samples were collected and spleens were harvested two weeks after the administration of the final immunisations. Serum samples were screened for anti-YFV antibodies by an enzyme-linked immunosorbent assay (ELISA) and immunofluorescence to determine the induction of a humoral immune response. Five of the twenty mice in groups immunised with the DNA-launched pSinED-lll replicon developed low level antibody responses illustrating the potential of the pSinED-lll replicon to induce a humoral immune response. Splenocytes were stimulated in cell culture with concanavalin A, YFV ED-lll protein, or no stimulant to facilitate the detection of cytokine release using ELISA’s. A predominantly T-helper 1 cell-mediated immune response characterised by high level release of interferon-γ post-stimulation with the YFV ED-lll protein in vitro was elicited. The release of interleukin-10 post-stimulation may be associated with the prevention of immunologically mediated damage to the host by preventing an overzealous inflammatory immune response. Antibody-mediated anti-vector immunity should not negatively impact the immunogenicity of the DNA-launched pSinED-lll replicon as antibody directed against the encoded Sindbis virus non-structural proteins was not detected in mouse sera. In conclusion, the pSinED-lll replicon was shown to have the potential to induce both a cell-mediated and a humoral immune response post-immunisation. However, optimisation of the humoral immune response will be required.en_ZA
dc.description.abstractAfrikaans: Geelkoorsvirus (GKV), ‘n muskiet-oordraagbare virus wat behoort tot die familie Flaviviridae en genus Flavivirus, is ‘n belangrike oorsaak van morbiditeit en mortaliteit in geelkoors endemiese gebiede, veral in Wes-Afrika. In die mens veroorsaak GKV, geelkoors, ‘n siekte wat gekenmerk word deur nierversaking, geelsug en/of bloeding. Die las van die siekte is die hoogste in Afrika waar ongeveerd 90% van aangemeldte gevalle wêreldwyd voorkom. Ten spyte van die beskikbaarheid van hoogs effektiewe lewend verswakte entstowwe teen GKV is die beraamde voorkoms van geelkoors in Afrika vir 2013 steeds 130 000 ernstige gevalle en 78 000 sterftes. Die beskikbare lewend verswakte entstowwe is teenaangedui vir gebruik in immuunonderdrukte pasiënte en individue met eier en/of hoenderallergieë. Entstof-verwante neurotropiese newe-effekte wat lei tot die ontwikkeling van meningoenkefalitis/breinvliesontsteking in babas en entstof-verwante viserotropiese newe-effekte wat lei tot siekte wat natuurlike infeksie met GKV naboots is aangemeld. Entstof-verwante viserotropiese newe-effekte word geassosieer met ‘n sterftekoers wat 40% oorskry. Daar is dus ‘n behoefte vir ‘n veiliger alternatief wat die gebruik van die beskikbare lewend verswakte entstowwe kan aanvul. Die doel van hierdie studie was die voorbereiding van ‘n DNS-geloodsde replikon teen GKV en om die immunogenisiteit van die DNS-geloodsde pSinED-lll replikon te bepaal wat insae sal verskaf tot die toepaslikheid van DNS-geloodsde replikons vir entstof ontwikkeling. Die pSinGFP replikon enkodeer die groen fluoresserende proteïen (GFP) en is goedkunstiglik deur Prof. Mark Heise verskaf. Die uitdrukking van die GFP is bevestig in geselekteerde soogdierselle na transfeksie met die pSinGFP replikon en gevolglik was die werking van die replikon elemente en die uitdrukking van die kodeerde proteïen bevestig. Die geen wat die GFP kodeer was verwyder uit die pSinGFP replikon en vervang met ‘n vervaardigde kodon-geoptimiseerde geen wat die GKV omhulsel domain lll (OD-lll) proteïen kodeer met behulp van gerigte klonering. Die nukleotiedvolgorde was bevestig deur tweerigting volgordebepaling in die gebied wat die ingevoegde geen bevat. Daarna was die uitdrukking van die kodeerde proteïen bevestig in geselekteerde soogdierselle. Proteïen uitdrukking was bevestig deur die opsporing van die C-terminale histidien merker met behulp van teen-His6 muis monoklonale teenliggame. Die uitgedrukte proteïen is daarna gekarateriseer deur immunofluoressensie met muis immuunserum wat voorheen bepaal is om teen-GKV OD-lll teenliggame te bevat. Reaktiwiteit van die uitgedrukte proteïen met teen-GKV OD-lll teenliggame het die gebruik van die DNS-geloodsde pSinOD-lll replikon vir ‘n muis immunisering studie bekragtig. Muise was binnespiers ingeënt volgens ‘n vooraf goedgekeurde immuniseringskema wat bestaan het uit slegs DNS, sowel as gemengde modale immuniseringskemas. Serum monsters was ingesamel en die milt was asepties verwyder twee weke na die toediening van die finale inentings. Teenliggame teen GKV was opgespoor in serum monsters met behulp van ‘n ensiem-gekoppelde immunosorbent toets (ELISA) en immunofluoressensie om te bepaal of ‘n humorale immuunrespons ontlok was. In 5/20 muise in groepe ingeënt met pSinOD-lll replikon DNS was ‘n lae vlak teenliggaam respons opgespoor wat die potensiaal van die pSinOD-lll replikon om ‘n humorale immuunrespons te ontlok illustreer. Om die opsporing van sitokiene met behulp van ‘n ELISA te fasiliteer was miltselle met konkanavalien A, GKV OD-lll proteïen of met geen stimulant gestimuleer in selkultuur. ‘n Oorwegend T-helper 1 sel-bemiddelde immuunrespons gekenmerk deur ‘n hoë vlak van interferon-γ vrystelling was bepaal na stimulasie met die GKV OD-lll proteïen in vitro. Die vrystelling van interleukin-10 na stimulasie kan moontlik geassosieer word met die voorkoming van immunologies bemiddelde skade aan die gasheer deur die voorkoming van ‘n opruiende inflammatoriese immuunrespons. Teenliggaam-bemiddelde teen-vektor immuniteit behoort nie die immunogenisiteit van die DNS-geloodsde pSinOD-lll replikon te affekteer nie, aangesien geen teenliggame teen die kodeerde Sindbis virus nie-strukturele proteïene opgespoor was in die serum van die muise nie. Ten slotte was die potensiaal van die DNS-geloodsde pSinOD-lll replikon om ‘n sel-bemiddelde en ‘n humorale immuunrespons na inenting te ontlok gedemonstreer. Die humorale immuunrespons wat ontlok was sal egter versterk moet word.af
dc.description.sponsorshipNational Health Laboratory Service Research Trusten_ZA
dc.description.sponsorshipNational Research Foundation (NRF)en_ZA
dc.description.sponsorshipPoliomyelitis Research Foundationen_ZA
dc.description.sponsorshipUniversity of the Free State, School of Medicineen_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectYellow fever virusen_ZA
dc.subjectEnvelope domain lll proteinen_ZA
dc.subjectDNA-launched vaccineen_ZA
dc.subjectReplicon-baseden_ZA
dc.subjectCell-mediateden_ZA
dc.subjectHumoralen_ZA
dc.subjectImmune responseen_ZA
dc.subjectDissertation (M.Med.Sc. (Medical Microbiology and Virology))--University of the Free State, 2014en_ZA
dc.subjectYellow fever -- Vaccinationen_ZA
dc.subjectImmunogeneticsen_ZA
dc.titlePreparation and immunogenicity of a candidate replicon based yellow fever vaccineen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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