Assessment and optimization of Theileria parva sporozoite fulllength p67 antigen expression in mammalian cells

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cg.authorship.typesNot CGIAR international instituteen_US
cg.contributor.affiliationUniversity of Parmaen_US
cg.contributor.affiliationWashington State Universityen_US
cg.contributor.crpLivestocken_US
cg.contributor.donorUnited States Agency for International Developmenten_US
cg.contributor.donorBill & Melinda Gates Foundationen_US
cg.howPublishedFormally Publisheden_US
cg.identifier.doihttps://doi.org/10.1371/journal.pntd.0005803en_US
cg.isijournalISI Journalen_US
cg.issn1935-2735en_US
cg.issue8en_US
cg.journalPLOS Neglected Tropical Diseasesen_US
cg.reviewStatusPeer Reviewen_US
cg.speciesTheileria parvaen_US
cg.subject.ilriANIMAL DISEASESen_US
cg.subject.ilriCATTLEen_US
cg.subject.ilriDISEASE CONTROLen_US
cg.subject.ilriECFen_US
cg.subject.ilriLIVESTOCKen_US
cg.subject.ilriVACCINESen_US
cg.volume11en_US
dc.contributor.authorTebaldi, G.en_US
dc.contributor.authorWilliams, L.B.en_US
dc.contributor.authorVerna, A.E.en_US
dc.contributor.authorMacchi, F.en_US
dc.contributor.authorFranceschi, V.en_US
dc.contributor.authorFry, Lindsay M.en_US
dc.contributor.authorKnowles, Donald P.en_US
dc.contributor.authorDonofrio, G.en_US
dc.date.accessioned2019-02-26T16:01:28Zen_US
dc.date.available2019-02-26T16:01:28Zen_US
dc.identifier.urihttps://hdl.handle.net/10568/99702en_US
dc.titleAssessment and optimization of Theileria parva sporozoite fulllength p67 antigen expression in mammalian cellsen_US
dcterms.abstractDelivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant versions of p67 derived from bacteria, insect, and mammalian cell systems are expressed in non-native and highly unstable forms. The stable expression of full-length recombinant p67 in mammalian cells has never been described and has remained especially elusive. In this study, p67 was expressed in human-derived cells as a full-length, membrane-linked protein and as a secreted form by omission of the putative transmembrane domain. The recombinant protein expressed in this system yielded primarily two products based on Western immunoblot analysis, including one at the expected size of 67 kDa, and one with a higher than expected molecular weight. Through treatment with PNGase F, our data indicate that the larger product of this mammalian cell-expressed recombinant p67 cannot be attributed to glycosylation. By increasing the denaturing conditions, we determined that the larger sized mammalian cell-expressed recombinant p67 product is likely a dimeric aggregate of the protein. Both forms of this recombinant p67 reacted with a monoclonal antibody to the p67 molecule, which reacts with the native sporozoite. Additionally, through this work we developed multiple mammalian cell lines, including both human and bovine-derived cell lines, transduced by a lentiviral vector, that are constitutively able to express a stable, secreted form of p67 for use in immunization, diagnostics, or in vitro assays. The recombinant p67 developed in this system is immunogenic in goats and cattle based on ELISA and flow cytometric analysis. The development of a mammalian cell system that expresses full-length p67 in a stable form as described here is expected to optimize p67-based immunization.en_US
dcterms.accessRightsOpen Accessen_US
dcterms.audienceScientistsen_US
dcterms.available2017-08-11en_US
dcterms.bibliographicCitationTebaldi, G., Williams, L.B., Verna, A.E., Macchi, F., Franceschi, V., Fry, L.M., Knowles, D.P. and Donofrio, G. 2017. Assessment and optimization of Theileria parva sporozoite fulllength p67 antigen expression in mammalian cells. PLoS Neglected Tropical Diseases 11(8): e0005803.en_US
dcterms.issued2017-08-11en_US
dcterms.languageenen_US
dcterms.licenseCC0-1.0en_US
dcterms.publisherPublic Library of Scienceen_US
dcterms.subjecttheileriaen_US
dcterms.subjectcattleen_US
dcterms.subjectanimal diseasesen_US
dcterms.subjectvaccinesen_US
dcterms.subjectdisease controlen_US
dcterms.subjecteast coast feveren_US
dcterms.typeJournal Articleen_US

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Improved vaccines for the control of East Coast fever in cattle in Africa
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