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Assessment and optimization of Theileria parva sporozoite full-length p67 antigen expression in mammalian cells.

ABSTRACT: Delivery 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.

SUBMITTER: Tebaldi G

PROVIDER: S-EPMC5568440 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Assessment and optimization of Theileria parva sporozoite full-length p67 antigen expression in mammalian cells.

Tebaldi Giulia G Williams Laura B LB Verna Andrea E AE Macchi Francesca F Franceschi Valentina V Fry Lindsay M LM Knowles Donald P DP Donofrio Gaetano G

PLoS neglected tropical diseases 20170811 8

Delivery 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 ...[more]

PMID: 28800590

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Project description:East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, is a leading cause of morbidity and mortality in cattle of sub-Saharan Africa. The infection and treatment method (ITM) is currently the only vaccine available to control T. parva. Although ITM elicits levels of protection, its widespread adoption is limited by costs, laborious production process, and antibiotic co-treatment requirement, necessitating the development of a more sustainable vaccine. To this end, efforts have been concentrated in the identification of new T. parva vaccine antigens and in the development of suitable platforms for antigen expression. In this study, we investigated the molecular and antigenic properties of T. parva antigen Tp9 expressed by mammalian cells. Data indicate that Tp9 contains a signal peptide that is weakly functional in mammalian cells. Thus, Tp9 secretion from mammalian cells increased 10-fold after the native signal peptide was replaced with the human tissue plasminogen activator signal peptide (tPA). Sera from all T. parva-immune cattle recognized this recombinant, secreted Tp9. Additionally, PBMC from ITM-immunized cattle produced significant (p < 0.05) amounts of IFNγ following ex vivo exposure to Tp9, but this response varied between cattle of different MHC class I and class II genotypes. In addition, depletion experiments demonstrated that IFNγ to Tp9 was primarily produced by CD4+ T cells. Molecular analysis demonstrated that Tp9 presents a signal peptide that is weakly functional in mammalian cells, suggesting that it remains within lymphocytes during infection. Tp9 secretion from mammalian cells was substantially increased when the tPA secretion signal sequence was substituted for the native secretion signal sequence. Using full-length, recombinant Tp9 secreted from mammalian cells, we demonstrated that T. parva-immune cattle develop both humoral and cellular immune responses to this antigen. Collectively, these results provide rationale for further evaluation of Tp9 as a component of a T. parva subunit vaccine.

Dataset Information

Assessment and optimization of Theileria parva sporozoite full-length p67 antigen expression in mammalian cells.

Publications

Assessment and optimization of Theileria parva sporozoite full-length p67 antigen expression in mammalian cells.

Similar Datasets

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