Targeted Surface Expression of an Exogenous Antigen in Stably Transfected Babesia bovis

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. One use of transfected B. bovis parasites may be as a vaccine delivery platform. 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 a selected antigen designed to be delivered to the surface of the parasites. The strategy for duplicating the number of transfected genes was based on the use of the putative bidirectional promoter of the B. bovis 1.4 Kb ef-1α 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-1α 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.

Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine

“…Babesia bovis T3B parasites were electroporated with plasmids pMSASignal-HlGST-GFP-BSD , and control plasmids pEf-msa-1-Bm86ep-gfp-bsd [39] or pBlueScript ( pBS ). Blasticidin resistant parasites electroporated with plasmid pEf-msa-Bm86ep-gfp-bsd , designated Tf-Bm86ep-gfp-bsd, or plasmid pMSASignal-HlGST-GFP-BSD , termed HlGST, emerged in in vitro cultures starting 16 days after electroporation (Fig 2A).…”

“…It was previously proposed that a transfected B . bovis expressing heterologous parasite proteins can be used as carriers to deliver selected antigens to the bovine immune system [39]. Clearly, transfection methods together with other related gene editing tools allow production of specifically designed strains for developing alternative and better defined attenuated B .…”

“…Ideally, such foreign antigen delivery platforms could be applied for developing dual Babesia and tick vaccines by producing a B . bovis strain able to synthetize a tick protein that induces anti-tick immune responses during cattle infection as well as the expected anti-babesia immune response [2,39]. However the ability of transfected B .…”

Identification of interchangeable cross-species function of elongation factor-1 alpha promoters in Babesia bigemina and Babesia bovis

“…A current limitation for the development of such gene analysis systems is the lack of defined B. bigemina promoters and a method to introduce foreign DNA into B. bigemina . The promoters controlling the expression of elongation factor 1-alpha ( ef-1α ) genes in apicomplexan parasites are generally regarded as strong constitutive promoters [12, 13]. In B. bovis the ef-1α includes an arrangement of two identical head to head genes separated by a 1.4 kb region containing two apparently distinct promoters [12, 13].…”

“…The promoters controlling the expression of elongation factor 1-alpha ( ef-1α ) genes in apicomplexan parasites are generally regarded as strong constitutive promoters [12, 13]. In B. bovis the ef-1α includes an arrangement of two identical head to head genes separated by a 1.4 kb region containing two apparently distinct promoters [12, 13]. Therefore a rational approach for the identification of B. bigemina promoters consists of searching for the presence of a similar structural and functional arrangement for the ef-1α locus in this parasite.…”