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

Authors: TEBALDI, GIULIA - University Of Parma; WILLIAMS, LAURA - Washington State University; VERNA, ANDREA - University Of Parma; MACCHI, FRANCESCA - University Of Parma; FRANCESHI, VALENTINA - University Of Parma; Fry, Lindsay; Knowles Jr, Donald; DONOFRIO, GAETANO - University Of Parma

Submitted to: PLOS Neglected Tropical Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/12/2017
Publication Date: 8/11/2017
Citation: Tebaldi, G., Williams, L.B., Verna, A.E., Macchi, F., Franceshi, V., Fry, L.M., Knowles Jr, D.P., Donofrio, G. 2017. Assessment and optimization of theileria parva sporozoite full-length p67 antigen expression in mammalian cells. PLOS Neglected Tropical Diseases. https://doi.org/10.1371/journal.pntd.0005803.

Interpretive Summary: Parasites that infect the blood of livestock present a significant challenge because they can cause health issues decreasing meat and fiber and animal that survive the initial infection are infected for life. Critically, animals that survive the initial infection are protected for life. The complexity of the parasites that cause diseases such as babesiosis or theileriosis has prevented development of safe and efficacious vaccines. These parasites of livestock are related to the organisms that cause malaria. The work described here is the first report of the ability to produce through molecular biology a full length copy of an important component of Theileria parva that has been shown to induce protective immunity. This component, referred to as p67 will now be tested as a vaccine for East Coast Fever, an important production limiting disease in cattle.

Technical 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 fulllength 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.