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. 2017 May 30;17(1):47.
doi: 10.1186/s12896-017-0370-5.

Development of plant-produced protein body vaccine candidates for bluetongue virus

Albertha R van Zyl  1 , Ann E Meyers  2 , Edward P Rybicki  1   3
Affiliations

Development of plant-produced protein body vaccine candidates for bluetongue virus

Albertha R van Zyl et al. BMC Biotechnol. .

Abstract

Background: Bluetongue is a disease of domestic and wild ruminants caused by bluetongue virus serotypes (BTV), which have caused serious outbreaks worldwide. Commercially available vaccines are live-attenuated or inactivated virus strains: these are effective, but there is the risk of reversion to virulence or reassortment with circulating strains for live virus, and residual live virus for the inactivated vaccines. The live-attenuated virus vaccines are not able to distinguish naturally infected animals from vaccinated animals (DIVA compliant). Recombinant vaccines are preferable to minimize the risks associated with these vaccines, and would also enable the development of candidate vaccines that are DIVA-compliant.

Results: In this study, two novel protein body (PB) plant-produced vaccines were developed, Zera®-VP2ep and Zera®-VP2. Zera®-VP2ep contained B-cell epitope sequences of multiple BTV serotypes and Zera®-VP2 contained the full-length BTV-8 VP2 codon-optimised sequence. In addition to fulfilling the DIVA requirement, Zera®-VP2ep was aimed at being multivalent with the ability to stimulate an immune response to several BTV serotypes. Both these candidate vaccines were successfully made in N. benthamiana via transient Agrobacterium-mediated expression, and in situ TEM analysis showed that the expressed proteins accumulated within the cytoplasm of plant cells in dense membrane-defined PBs. The peptide sequences included in Zera®-VP2ep contained epitopes that bound antibodies produced against native VP2. Preliminary murine immunogenicity studies showed that the PB vaccine candidates elicited anti-VP2 immune responses in mice without the use of adjuvant.

Conclusions: These proof of concept results demonstrate that Zera®-VP2ep and Zera®-VP2 have potential as BTV vaccines and their development should be further investigated.

Keywords: Bluetongue virus; Nicotiana benthamiana; Protein body; Vaccine; Zera®.

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Figures

Fig. 1
Fig. 1
Construction of VP2ep. a Selected regions of the multiple alignment of 8 selected BTV VP2 serotype amino acid sequences are shown. The predicted epitope regions corresponding to most of the serotypes are shown below the alignment in bold and boxed in blue and green. The homologous region is boxed in purple. b The putative amino acid and nucleotide sequences of VP2ep
Fig. 2
Fig. 2
Expression of Zera®-VP2ep and Zera®-VP2 in N. benthamiana. Western blots of crude leaf extracts from leaves infiltrated with Zera®-VP2ep (a) and Zera®-VP2 (b) constructs at infiltration OD600 values of 0.5 and 1.5, respectively and harvested at 2, 3 5 and 7 days post infiltration (dpi). Expressed protein was detected with α-VP2R polyclonal antibody. Samples from negative control plants (−) were infiltrated with infiltration medium only. Red arrows indicate the position of the appropriately-sized expressed proteins. Lanes M represent the molecular weight marker with sizes indicated in kDa. c N. benthamiana leaves infiltrated with different A. tumefaciens cell concentrations. Leaves expressing Zera®-VP2ep and Zera®-VP2 were photographed at 3 and 7 dpi, respectively
Fig. 3
Fig. 3
Transmission electron micrographs of protein bodies. Leaf sections were infiltrated with a pEAQ-HTZera®-VP2ep and b pEAQ-HTZera®-VP2 and c infiltration medium as a negative control. PBs for both Zera®-VP2ep (a – blue arrows) and Zera®-VP2 (b – pink arrows) were present as electron-dense structures within the cytoplasm of the infiltrated leaves. No similar structures were present in the negative control samples (c). Scale bars: a and b: 0.5 μm; c: 0.2 μm. CW: cell wall, CPT: chloroplast, CYT: cytoplasm, ER: endoplasmic reticulum
Fig. 4
Fig. 4
Purification of PBs. a Western blot analysis of crude Zera®-VP2ep (i) and Zera®-VP2 (ii) extracted protein using α-VP2R as primary antibody. White arrows indicate the respective proteins with the black arrow showing an oligomerised fusion of VP2ep. In both cases the negative control (−) was plant material infiltrated with infiltration medium only that was extracted using the same method. Lanes M indicate the molecular weight marker in kDa. b α-VP2R dot blots of (i) the negative control, (ii) Zera®-VP2ep and (iii) Zera®-VP2 purified using a 42% sucrose cushion
Fig. 5
Fig. 5
Analysis of serum from immunised mice. a Titration of the mouse antisera produced Zera®-VP2ep (blue line), Zera®-VP2 (pink line) and DPBS (negative control, grey line) vaccine candidates as well as titration of positive control sheep serum produced against BTV-8 VLPs [20] to validate the indirect ELISA (b). The markers indicate the mean value of triplicate samples from both animal experiments, and error bars indicate the standard deviation. (c) Western blot detection of the E. coli-expressed VP2 fusion protein with 1:100 dilution of pooled mice sera from animals vaccinated with the Zera®-VP2ep and Zera®-VP2 vaccines. Lane M represents the molecular weight marker. The negative control (−) was performed with no primary antibody and sheep serum obtained from BTV-8 VLP vaccinated sheep [20] was used as the positive control (+). Lanes PB and FB represent the pre-and final bleed sera respectively. The white arrow indicates the E. coli-expressed VP2 fusion protein at ~163 kDa. PB – prebleed; FB – final bleed
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