|LAUGHERY, JM - Washington State University|
|Knowles Jr, Donald|
|MCELWAIN, TF - Washington State University|
Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/25/2014
Publication Date: 5/19/2014
Citation: Laughery, J., Knowles Jr, D.P., Schneider, D.A., Mcelwain, T., Suarez, C.E. 2014. Targeted surface expression of an exogenous antigen in stably transfected babesia bovis. PLoS One. DOI:10.1371/journal.pone.0097890.
Interpretive Summary: Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. Transfection allows for the expression of foreign genes in targeted cells. A goal of our lab is the production of Babesia bovis vaccine strains that can also elicit protection against its tick vector. In this manuscript we describe improvements on the Babesia bovis transfection system allowing co-expression of two distinct exogenous genes in transfected B. bovis. One of the transfected genes encodes for protective antigenic determinants, or "B-cell epitopes", that are present in effective anti-tick vaccines based on the Rhiphicephalus microplus tick Bm86 vaccine. Importantly, the new system is designed for expression of the Bm86 protective vaccine epitopes in the surface of the parasite, a feature designed to increase antigenic properties. Expression of the Bm86 epitopes on the surface of transfected merozoites was demonstrated using immunofluorescence analyses. These new developments advance the potential of transfected B. bovis as a future vaccine delivery platform.
Technical Abstract: Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. Here we propose using transfected B. bovis as an antigen delivery system. Previous transfection methods for B. bovis were limited by single expression sites and intracellular expression of transfected antigens. This study describes a novel transfection system in which two exogenous genes are expressed: one for selection and the other for antigen delivery to the surface of the parasites, a feature designed to increase antigenic properties. The strategy for duplicating the number of transfected genes was based on the use of the putative bidirectional promoter of the B. bovis 1.4kb ef-1 alpha intergenic region. The ability of this region to regulate two independent expression sites was demonstrated using a luciferase assay on transiently transfected B. bovis parasites and then incorporated into a stable transfection plasmid to control independent expression of the selectable marker GFP-BSD and another gene of interest. A chimeric gene was synthetized using sequences from the protective B-cell epitopes of Rhipicephalus microplus tick antigen Bm86 along with sequences from the surface exposed B. bovis major surface antigen-1. This chimeric gene was then cloned into the additional expression site of the transfection plasmid. Transfection of the B. bovis Mo7 strain with this plasmid resulted in stable insertion into the ef-1alpha locus and simultaneous expression of both exogenous genes. Expression of the Bm86 epitopes on the surface of transfected merozoites was demonstrated using immunofluorescence analyses. The ability to independently express multiple genes by the inclusion of a bidirectional promoter and the achievement of surface expression of foreign epitopes advances the potential of transfected B. bovis as a future vaccine delivery platform.