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

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cg.authorship.typesNot CGIAR international instituteen
cg.contributor.affiliationUniversity of Parmaen
cg.contributor.affiliationWashington State Universityen
cg.contributor.crpLivestock
cg.contributor.donorUnited States Agency for International Developmenten
cg.contributor.donorBill & Melinda Gates Foundationen
cg.howPublishedFormally Publisheden
cg.identifier.doihttps://doi.org/10.1371/journal.pntd.0005803en
cg.isijournalISI Journalen
cg.issn1935-2735en
cg.issue8en
cg.journalPLOS Neglected Tropical Diseasesen
cg.reviewStatusPeer Reviewen
cg.speciesTheileria parvaen
cg.subject.ilriANIMAL DISEASESen
cg.subject.ilriCATTLEen
cg.subject.ilriDISEASE CONTROLen
cg.subject.ilriECFen
cg.subject.ilriLIVESTOCKen
cg.subject.ilriVACCINESen
cg.volume11en
dc.contributor.authorTebaldi, G.en
dc.contributor.authorWilliams, L.B.en
dc.contributor.authorVerna, A.E.en
dc.contributor.authorMacchi, F.en
dc.contributor.authorFranceschi, V.en
dc.contributor.authorFry, Lindsay M.en
dc.contributor.authorKnowles, Donald P.en
dc.contributor.authorDonofrio, G.en
dc.date.accessioned2019-02-26T16:01:28Zen
dc.date.available2019-02-26T16:01:28Zen
dc.identifier.urihttps://hdl.handle.net/10568/99702
dc.titleAssessment and optimization of Theileria parva sporozoite fulllength p67 antigen expression in mammalian cellsen
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
dcterms.accessRightsOpen Access
dcterms.audienceScientistsen
dcterms.available2017-08-11
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
dcterms.issued2017-08-11
dcterms.languageen
dcterms.licenseCC0-1.0
dcterms.publisherPublic Library of Scienceen
dcterms.subjecttheileriaen
dcterms.subjectcattleen
dcterms.subjectanimal diseasesen
dcterms.subjectvaccinesen
dcterms.subjectdisease controlen
dcterms.subjecteast coast feveren
dcterms.typeJournal Article

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Improved vaccines for the control of East Coast fever in cattle in Africa
CRP Livestock journal articles