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dc.contributor.advisorBurt, F. J.
dc.contributor.authorSmouse, Shannon Lucrecia
dc.date.accessioned2015-11-25T08:23:08Z
dc.date.available2015-11-25T08:23:08Z
dc.date.issued2013-02
dc.identifier.urihttp://hdl.handle.net/11660/1904
dc.description.abstractEnglish: Yellow fever virus (YFV) virus is an arthropod-borne virus that causes viral hemorrhagic fever in humans in the tropical parts of both Africa and South America. The virus belongs to the family Flaviviridae, of the genus Flavivirus comprising of approximately 70 viruses. It is transmitted to vertebrates by the bite of an infected female mosquito, primarily the Aedes species. It is a re-emerging pathogen with case-fatality rates that can exceed 50% in humans. YFV can cause an acute febrile illness in humans which can progress to severe disease with hepatic and renal failure. The diagnosis of infection and testing of the immune status of vaccinees require reagents that are prepared in biosafety level (BSL) three and four facilities. Therefore the development of a recombinant antigen that does not require BSL three facilities for preparation and is safe to use, would have an important role in a diagnostic laboratory for detecting antibodies in infected individuals and vaccinees. Despite the availability of a live-attenuated efficacious vaccine, it is not recommended for immunocompromised individuals, thus development of new generation vaccines would have important public health implications. Identification and mapping of antigenic regions and viral epitopes is important for development of subunit vaccines and improved diagnostics. Subunit vaccines focusing on antigens that induce a protective immune response provide a safe approach to the development of vaccines against diseases causing severe and frequently fatal haemorrhagic fevers. The aim of this study was to identify immunodominant viral proteins that induce detectable antibody responses that could be used for developing diagnostic assays and to identify linear B cell epitopes on selected viral proteins. The complete open reading frame of the genes encoding the domain III (EDIII) region of the envelope protein, capsid (C) and NS4a proteins of YFV were amplified, from the 17D strain of YFV, by RT-PCR using primers specifically designed from sequence data retrieved from GenBank. Oligonucleotide primers were modified with BamHI and HindIII restriction enzyme sites that facilitated downstream cloning. Each amplicon was cloned into the pGEM®-T Easy cloning vector using T/A cloning. Each gene was rescued from the recombinant plasmid using BamHI and HindIII restriction enzyme sites and ligated into bacterial expression system, pQE-80L vector. In a previous study, the YFV EDIII gene was cloned into pQE-80L and expressed in JM109 Escherichia coli cells however extremely low yields were obtained. In this study the expression levels were improved using different cell lines and optimizing incubation conditions. An insoluble 13 kDa protein was expressed from the construct and confirmed by Western blot analysis. The protein was expressed with a 6 x Histidine tag that was used to facilitate purification using a Ni2+ column under denaturing conditions. Attempts to express the YFV C and NS4a proteins were not successful and expression was abandoned. In an attempt to improve solubility the YFV EDIII gene was excised from the pGEM®-T Easy vector and subsequently cloned into pCold TF bacterial expression vector. A ~65 kDa soluble protein was expressed from the construct and purified under native conditions. The functional activity of the recombinant antigens in ELISA was compared with whole cell lysate antigen prepared from cell cultures infected with YFV. The biological activity of the recombinant YFV pQE-80L-EDIII antigen was confirmed in immunoassays using serum samples from humans vaccinated with YFV vaccine. Positive sera failed to react in ELISA using pCold TF expressed antigen and this antigen was excluded from further assays. A total of 20/24 serum samples from human vaccinees collected at varying stages after vaccination reacted in an ELISA with the recombinant YFV pQE-80L-EDIII protein and 24/24 reacted in ELISA with whole cell lysate antigen. The EDIII region of the envelope protein was shown to be able to differentiate between West Nile Virus infection and YFV infection in a limited number of convalescent horse sera. The recombinant EDIII protein was used to immunize mice. Serum samples collected from the mice reacted against whole cell lysate antigen in ELISA and was shown to have neutralising antibodies using an in vitro neutralisation assay. Hence the EDIII region of the envelope protein likely induces an important protective immune response. Finally, bioinformatics was used to predict possible epitope regions and using peptide libraries spanning predicted sites, one potential epitopic region was identified in the EDIII protein. Putative epitopic and antigenic regions along the length of the C, NS4a and EDIII proteins of each strain were predicted using the BCPREDS and ABCpred software. In conclusion, the EDIII protein, an immunodominant antigen of YFV, prepared in this study has some potential for differentiation of flavivirus antibodies although it lacks sensitivity for routine diagnosis. A potential epitope, TGHGTVVMQ, from amino acid 21 to 29 on the EDIII protein was identified using bioinformatics and was shown to have reactivity against immune sera. The significance of this epitope needs further investigation. Finally the EDIII region of the YFV protein shows potential as a target region for vaccine development as shown for other flaviviruses but which has not previously been published for YFV.en_ZA
dc.description.abstractAfrikaans: Die Geelkoors virus is ‘n virus wat deur geleedpotiges gedra word en wat virale haemoragiese koors veroorsaak in mense in die tropiese gebiede van beide Afrika en Suid-Amerika. Die virus is lid van die familie Flaviviridae, van die genus Flavivirus wat ongeveer 70 virusse insluit. Dit word na gewerwelde diere oorgedra deur die byt van ‘n geïnfekteerde vroulike muskiet van hoofsaaklik die Aedes spesie. Dit is ‘n patogeen wat weer opnuut sy verskyning gemaak het en wat ‘n mortaliteit van meer as 50% in menslike gevalle het. Geelkoors kan ‘n akute siekte in mense veroorsaak, met gepaardgaande koors wat kan vererger tot ernstige siekte met lewer en nierversaking. Die diagnose van infeksie en toets van die immuunstatus van ingeënte mense vereis reagense wat in bioveiligheidsvlak (BSL) drie en vier fasiliteite voorberei moet word. Die ontwikkeling van ‘n rekombinante antigeen, wat nie BSL drie fasiliteite vir voorbereiding vereis nie, sal dus ‘n belangrike rol speel in ‘n diagnostiese laboratorium vir die opsporing van antiliggaampies in geïnfekteerde individue en geïmmuniseerde mense. Ondanks die beskikbaarheid van ‘n lewende-geattenueerde effektiewe entstof, word dit nie aanbeveel vir immuunonderdrukte mense nie en sal die ontwikkeling van ‘n nuwe generasie entstof belangrike implikasies vir publieke gesondheid inhou. Identifisering en kartering van antigeniese gedeeltes en virale epitope is belangrik vir die ontwikkeling van subeenheid entstowwe en verbeterde diagnose. Subeenheid entstowwe wat fokus op antigene wat ‘n beskermende immuunreaksie induseer bied ‘n veilige benadering tot die ontwikkeling van entstowwe teen siektes wat ernstige en gereeld dodelike haemoragiese koors veroorsaak. Die doel van hierdie studie was die identifisering van immuundominante virale proteïene wat opspoorbare antigeniese reaksies veroorsaak wat gebruik kon word vir die ontwikkeling van diagnostiese toetse en om liniêre B sel epitope op geselekteerde virale proteïene te identifiseer. Die volledige oop leesraam van die gene wat kodeer vir die domein III gedeelte van die omhulsel proteïen, kapsel en NS4a proteïene van geelkoors, is geamplifiseer vanaf die 17D stam van geelkoors deur middel van RT-PKR en die gebruik van priemstukke wat spesifiek ontwerp is vanaf basispaarfragmentdata wat van GenBank afgetrek is. Oligonukleotied priemstukke is gemodifiseer met BamHI and HindHII beperkingsensiem posisies wat “downstream” kloning fasiliteer. Elke amplikon is in ‘n pGEM®-T Easy cloning vektor gekloneer deur gebruik van T/A klonering. Elke geen is vanuit die rekombinante plasmied herwin deur gebruik van die BamHI en HindIII beperkingsensiem posisies en ingebou in ‘n bakteriese uitdrukkingssisteem vektor, pQE-80L. In ‘n vorige studie is die geelkoors EDIII geen in pQE-80L gekloneer en uitgedruk in JM109 Escherichia coli selle, maar ‘n uitermate lae opbrengs is verkry. In hierdie studie is die vlakke van uitdrukking verhoog deur die gebruik van verskillende sellyne en optimisasie van die inkubasie kondisies. ‘n Onoplosbare 13 kDa proteïen is uitgedruk met behulp van die konstruk en bevestig deur Westelike blot analise. Die proteïen is uitgedruk met ‘n 6 x Histidine merker wat gebruik is om suiwering te fasiliteer deur gebruik van ‘n Ni2+ kolom onder denaturerings kondisies. Pogings om die geelkoors C en NS4a proteïene uit te druk was nie suksesvol nie en is gestaak. In ‘n poging om die oplosbaarheid te verbeter is die geelkoors EDIII geen uitgesny uit die pGEM®-T Easy vektor en daarna in ‘n pCold TF bakteriese vektor gekloneer. ‘n ~65 kDa oplosbare proteïen is met behulp van die konstruk uitgedruk en gesuiwer onder natuurlike toestande. Die funksionele aktiwiteit van die rekombinante antigene in ELISA is vergelyk met heelsel lisaat antigeen wat voorberei is vanaf selle wat met geelkoors geïnfekteer is. Die biologiese aktiwiteit van die rekombinante geelkoors pQE-80L-EDIII antigeen is bevestig in immuuntoetse deur gebruik van serum monsters van mense wat met geelkoors ingeënt is. Positiewe sera wou nie in ELISA reageer wanneer pCold TF uitdrukkings antigeen gebruik is nie en hierdie antigeen is uitgelaat uit verdere toetse. ‘n Totaal van 20/24 serum monsters van geënte mense tydens verskillende stadiums na inenting, het gereageer in ‘n ELISA met die rekombinante geelkoors pQE-80L-EDIII proteïen en 24/24 het in ELISA gereageer met heelsel lisaat antigeen. Dit kon aangetoon word dat die EDIII gedeelte van die omhulsel proteïen in staat was om tussen West Nile Virus infeksie en geelkoors infeksie te onderskei in ‘n beperkte aantal sera van herstelde perde. Die rekombinante EDIII proteïen is gebruik om muise in te ent. Serum monsters wat van die muise versamel is, het reageer teen heelsel lisaat antigeen in ELISA en dit is aangetoon dat die muise neutraliserende antiliggame het, in ‘n in vitro neutralisasie toets. Die EDIII gedeelte van die E proteïen het dus waarskynlik ‘n belangrike beskermende immuunrespons uitgelok. Laastens is bioinformatika gebruik om moontlike epitoop gedeeltes te voorspel en deur peptiedbiblioteke te gebruik van al die voorspelde gedeeltes, kon een potensiële epitoop gedeelte in die EDIII proteïen geïdentifiseer word. Voorspelde epitopiese en antigeniese gedeeltes oor die lengte van die kapsied, NS4a en EDIII proteïene van elke stam is voorspel deur gebruik van die BCPREDS en ABCpred sagteware. Ter afsluiting. Die EDIII proteïen, ‘n immuundominante antigeen van geelkoors, wat in hierdie studie voorberei is, het effense potensiaal vir die onderskeiding van flavivirus antiliggaampies alhoewel dit nie sensitief genoeg is vir roetine diagnose nie. ‘n Potensiële epitoop, TGHGTVVMQ, van aminosuur 21 tot 29 op die EDIII proteïen is geïdentifiseer en deur gebruik van bioinformatika is aangetoon dat dit reaktiwiteit teen immuun sera het. Die belang van hierdie epitoop moet egter verder bestudeer word. Uiteindelik toon die EDIII gedeelte van die geelkoors proteïen potensiaal as ‘n teiken gedeelte vir entstof ontwikkeling soos aangetoon vir ander flavivirusse, maar wat nog nie voorheen vir geelkoors gepubliseer is nie.aaf
dc.description.sponsorshipGrow Our Own Timber (GOOT) Scholarshipen_ZA
dc.description.sponsorshipStars of Academe and Research (SoAR) Scholarshipen_ZA
dc.description.sponsorshipPostgraduate School of Medicine Research Councilen_ZA
dc.description.sponsorshipNational Health Laboratory Service (NHLS) Research Trusten_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectFlavivirusesen_ZA
dc.subjectYellow feveren_ZA
dc.subjectYellow fever -- Vaccinationen_ZA
dc.subjectAntigensen_ZA
dc.subjectDissertation (M.Med.Sc. (Medical Microbiology and Virology))--University of the Free State, 2013en_ZA
dc.titleIdentification of antigenic regions and linear B cell epitopes on yellow fever virusen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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