Preparation and immunogenicity of a candidate replicon based yellow fever vaccine

English: 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 clon
Afrikaans: 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
National Health Laboratory Service Research Trust
National Research Foundation (NRF)
Poliomyelitis Research Foundation
University of the Free State, School of Medicine