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

Abstract

The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein-blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves. Collectively, these data show the efficacy of a transfected HlGST-Cln B. bovis parasite to induce detectable anti-glutathione-S-transferase antibodies and a reduction in tick size and fecundity of R. microplus feeding in experimentally inoculated animals.

Fig 1. Map of the Ef-1α gene structure and the pMSASignal-HlGST-GFP-BSD plasmid.

The bidirectional promoter and orfs of Ef1α-A and B are represented in the upper part of the panel. The dotted lines indicate the targeted site for insertion of the transfected sequences into the genome of the B. bovis. Arrows indicate the direction of transcription. The location of restriction sites of interest are also described in the figure.

Fig 2. Characterization of transfected parasites.

Two lines of transfected parasites HlGST1 and HlGST2 were generated by transfection of the T3B strain of B. bovis with plasmid pMSASignal-HlGST-GFP-BSD and analyzed in these experiments A) Comparison of the growth curves of non-transfected, control transfected (negative control electroporated with plasmid pBS, and unrelated positive control electroporated with plasmid pEf-msa-1-Bm86ep-gfp-bsd), and two lines of parasites electroporated with pMSASignal-HlGST-GFP-BSD (HlGST1 and HlGST2) after electroporation in the presence inhibitory doses of blasticidin. Blasticidin resistant parasites emerge ~16 days after the onset of selection only in the wells containing parasites electroporated with the pMSASignal-HlGST-GFP-BSD and pEf-msa-1-Bm86ep-gfp-bsd plasmids. B) Fluorescence microscopy of transfected parasites of the HlGST line (HlGST1 and 2, Upper panels), control GFP-B. bovis line (Unrelated) and non-transfected parasites (Lower panels).

Fig 3. HlGST expression in transfected parasites.

A) RT-PCR to detect transcripts of GFP-BSD, GST, and RAP as constitutive control. Lane 1: HlGST1 transfected B. bovis. Lane 2: HlGST2 transfected B. bovis. Lane 3: unrelated (GFP) transfected control B. bovis. Lane 4: non-transfected control B. bovis. Lane 5: MSASignal-HlGST-GFP-BSD plasmid. Lane 6: unrelated transfection control plasmid. Lane 7: negative control. B) Western Blot analysis on transfected parasites using αGST and αMSA-1 antibodies. Lane1: HlGST1 transfected B. bovis. Lane2: HlGST2 transfected B. bovis. Lane 3: unrelated transfected control B. bovis. Lane 4: unrelated (GFP) transfected control B. bovis. Lane 5: non-transfected control B. bovis.

Fig 4. Integration PCR analysis.

Upper panel: Representation of the genome area including the transfected genes integrated into the genome of HlGST-Cln B. bovis. The localization of the regions hybridizing with the primers used in PCR is represented in the map by arrows. Primers were used for the amplification of EF-GST, HlGST, RAP-1, GFP-EF and GFP/BSD. Lower Panel: Agarose gel analysis of the PCR amplification products: Lane 1: HlGST 1 transfected B. bovis line; lane 2: HlGST 2 transfected B. bovis; lane 3: unrelated (GFP) transfected control B. bovis; lane 4: non-transfected B. bovis: lane 5: MSASignal-HlGST-GFP-BSD plasmid; lane 6: unrelated transfection control plasmid; lane 7: negative no DNA control.

Fig 5. Analysis of the B. bovis transfected clonal lines.

Panel A: RT-PCR amplifications designed for the detection of HlGST and RAP transcripts. A single clonal line (#9) termed HlGST-cln, was able to produce both GST and RAP transcripts. Line 1 to 8: B. bovis cloned strains, -: negative control, +: positive control. B) Western blot using rabbit serum anti-HlGST to confirm HlGST expression by cloned parasites, confirming the presence of HlGST expression by cell line HlGST-cln (#9) (Red box). Line 1 to 8: cloned B. bovis strains, W: not transfected parasites, U: unrelated control, +: positive control with recombinant protein produced in E. coli. C) Southern blot analysis performed on B. bovis gDNA extracted from HlGST-Cln, HlGST and GFP-Cln B. bovis. Line 1: GST clonal strain; line 2: GST parent (mixed) population; line 3: GFP control strain; line 4: not transfected parasites; line 5: MSASignal-HlGST-GFP-BSD plasmid; line 6: GFP control plasmid. The arrows marked 1, 2, 3 and 4 represent the distinct hybridizing fragments identified. These fragments are graphically represented in the lower part of the panel C. Each fragment is described in a simplified map of the sequence, and an identifying number on their sides. The parallel bars showed on the sides of each fragment map represent the region digested by BglII. Lines under the maps in panel C represent the probes used, and the site of binding of the probe on the tested DNA. The dotted line represents the GST probe, the dashed line the GFP-BSD probe, and the continuous line, the EF probe.

Fig 6. HlGST parasites immunofluorescence.

Immunofluorescence assays using DAPI stained permeabilized or non-permeabilized free merozoites derived the from HIGST-Cln B. bovis cell line. Non-permeabilized free merozoites cells were incubated with anti-MSA-1 (Alexa Fluor 488) and anti-HlGST (Alexa Fluor 555). Non-permeabilized free merozoites were also incubated with anti-GFP (Alexa Fluor 488) and anti-HlGST (Alexa Fluor 555).Permeabilized merozoites were incubated with anti-GFP (Alexa Fluor 488) and anti-GST (Alexa Fluor 555), pre-immune rabbit serum (Alexa fluor 488), control anti-Tryp unrelated (Alexa Fluor 555). Columns represent DAPI, green (488nm), red (555nm) and green/red merged (488nm+555nm). The size bar is indicated on lower right image.

Fig 7. Infection of animals with clonal parasites.

Panel A: Daily clinical parameters (PCV and Rectal temperature) of the experimentally infected calves b1, b2 and b3. The dotted line in temperature graphic represents the threshold that indicate fever. Panel B: RAP-1 PCR amplification performed on daily total gDNA samples generated from blood of calves b1, b2 and b3. The expected 387 bp PCR fragment of the rap-1 gene using DNA isolated from washed RBC of infected animals is marked by arrows. The numbering over the lanes represent the day of blood collection after animals immunization. Size markers are shown on the left ends of the figures.

Fig 8. Detection of antibodies in calves experimentally infected with HlGST-Cln and GFP-cln parasites by cELISA and western blot analysis.

(A): Western blot analysis of recombinant HlGST incubated pre-immune and immune (12 and 56 days post immunization) sera from calves’ b1, b2, and b3 diluted 1:10. +: positive control, recombinant HlGST incubated with anti-HlGST serum, in a 1:1000 dilution. (B) Kinetics of antibody detection of the rhoptry-associated protein 1 (RAP-1) of B. bovis by cELISA. Samples obtained from each animal before and 10 days after experimental intravenous inoculation of the parasites. The threshold of inhibition is 21%, above which samples are considered positive is represented by the dashed line.