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Antibodies to in silico selected GPI-anchored Theileria parva proteins neutralize sporozoite infection in vitro.

ABSTRACT: East Coast fever (ECF) caused by Theileria parva kills cattle in East, Central and Southern Africa leading to significant economic losses. Vaccination is used as a control strategy against ECF and is presently dependent on deliberate infection with live sporozoites and simultaneous treatment with a long-acting oxytetracycline. Although effective, this method has serious limitations; the immunity is parasite strain specific and immunized cattle can become life-long asymptomatic carriers of the parasite, posing risk for the spread of the disease. In efforts to develop a subunit vaccine, the role of antibodies in the neutralization of T. parva sporozoites infection of host cells has been investigated and a circumsporozoite protein, p67, is able to induce such neutralizing antibodies. However, the p67 protein only protects a proportion of immunized cattle against T. parva challenge and such protection might be improved by inclusion of additional parasite antigens that neutralize sporozoite infection. In an attempt to identify such antigens, we searched the re-annotated T. parva genome for genes predicted to contain GPI anchor signals, since they are likely to be located on the cell surface, and expressed fragments of six of the selected genes in E. coli. The recombinant proteins were used to raise antisera in mice. Antisera to two proteins, TpMuguga_01g00876 and TpMuguga_01g00939, neutralized sporozoite infectivity to a high degree, while antisera to two additional proteins, TpMuguga_01g00095 and TpMuguga_04g00437, exhibited moderate neutralizing capacity. We conclude that these four antigens are potential vaccine candidates, which should be evaluated further in cattle.

SUBMITTER: Nyagwange J

PROVIDER: S-EPMC5956992 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Antibodies to in silico selected GPI-anchored Theileria parva proteins neutralize sporozoite infection in vitro.

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