Boosting in planta production of antigens derived from the porcine reproductive and respiratory syndrome virus (PRRSV) and subsequent evaluation of their immunogenicity

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is a disease of swine, caused by an arterivirus, the PRRS virus (PRRSV). This virus infects pigs worldwide and causes huge economic losses. Due to genetic drift, current vaccines are losing their power. Adaptable vaccines could provide a solution to this problem. This study aims at producing in planta a set of antigens derived from the PRRSV glycoproteins (GPs) to be included in a subunit vaccine. We selected the GP3, GP4 and GP5 and optimized these for production in an Arabidopsis seed platform by removing transmembrane domains (Tm) and/or adding stabilizing protein domains, such as the green fluorescent protein (GFP) and immunoglobulin (IgG) 'Fragment crystallizable' (Fc) chains. Accumulation of the GPs with and without Tm was low, reaching no more than 0.10% of total soluble protein (TSP) in homozygous seed. However, addition of stabilizing domains boosted accumulation up to a maximum of 2.74% of TSP when GFP was used, and albeit less effectively, also the Fc chains of the porcine IgG3 and murine IgG2a increased antigen accumulation, to 0.96% and 1.81% of TSP respectively, while the murine IgG3 Fc chain did not. Antigens with Tm were less susceptible to these manipulations to increase yield. All antigens were produced in the endoplasmic reticulum and accordingly, they carried high-mannose N-glycans. The immunogenicity of several of those antigens was assessed and we show that vaccination with purified antigens did elicit the production of antibodies with virus neutralizing activity in mice but not in pigs.

Figure 1. Schematic representation of the T-DNA bearing the seed-specific expression cassette (A), the PRRSV glycoproteins selected to be included in a vaccine (B) and the antigen formats derived thereof (C).

(A) The T-DNA carrying the neomycin phosphotransferase II gene which confers resistance to the antibiotic kanamycin and the cassette for high seed-specific expression driven by the β-phaseolin promoter, the leader sequence of the tobacco mosaic virus and the arcelin-5I terminator. The cassette is further supplemented with sequences encoding an ER-targeting and retention signal. (B) The full-length PRRSV glycoproteins used in the current study and supplemented with a His₆-tag (purple rod). The jagged line depicts the Tm. The full-length GP3 is placed between brackets as it was never produced as such. Only the GP3 without Tm was expressed, depicted as GP3-Tm in (C). (C) Overview of the manipulations performed on the GPs and the resulting set of antigenic proteins whose coding sequences were cloned into the seed-specific expression cassette. Abbreviations and symbols used: LB and RB, T-DNA left and right border, respectively; 3′ocs, 3′end of the octopine synthase gene; nptII, the neomycin phosphotransferase II gene; Pnos, promoter of the nopaline synthase gene; Pphas, promoter of the β-phaseolin gene; Ω, leader sequence of the ‘tobacco mosaic virus’; SS, sequence coding for the signal peptide of the 2S Arabidopsis seed storage protein; GOI, gene of interest; KDEL, coding sequence of the ER-retention signal; 3′Arc, terminator of the arceline5-I gene; Tm, transmembrane domain, jagged line; His₆-tag, immobilized metal affinity chromatography tag, purple rod; pFc, porcine IgG3 Fc, chain of blue ovals; mFc2a, murine IgG2a Fc, chain of orange ovals; mFc3, murine IgG3 Fc, chain of yellow ovals; GFP, green fluorescent protein, bright green oval.

Figure 2. Characterization of the GP3, GP4 and GP5 antigens.

Seed extracts of homozygous lines (unless stated differently) producing the different antigen formats were analyzed by western blotting under reducing conditions. Sixty µg of TSP was loaded. (A) The GP3 antigens detected with the α-GP3 mAb VII2D . (B) The GP4 antigens detected with the α-GP4 mAb XVI 11C . For the sGP4-Tm, sGP4-Tm:pFc and the GFP:GP4, segregating T2 stocks were analyzed and for the GFP:GP4-Tm and GP4-Tm:mFc2a, only 15 µg of TSP was loaded. (C) The GP5 antigens detected with the α-GP5 mAb II5A (left panel) or an antibody against the His₆-tag (right panel). For the GP5-Tm:pFc, a T2 segregating stock was analyzed. Abbreviations used: WT, wild-type seed extract; kDa, kilodalton.

Figure 3. Glycan analysis of the GP3, GP4 and GP5 antigens.

Seed extracts from homozygous lines (unless stated differently) producing the different antigen formats were treated with endoH or PNGaseF and analyzed by western blotting under reducing conditions. Thirty µg of TSP was loaded. (A) Glycan analysis of the GP3 antigens detected with the α-GP3 mAb VII2D . (B) Glycan analysis of the GP4 antigens detected with the α-GP4 mAb XVI 11c . For the sGP4-Tm, sGP4-Tm:pFc and GFP:GP4, a segregating T2 stock was used. (C) Glycan analysis of the GP5 antigens detected with an antibody against the His₆-tag. For the GP5-Tm:pFc, a segregating T2 stock was analyzed. Abbreviations used: -, no treatment; E, endoH treatment; P, PNGaseF treatment: WT, non-treated wild-type seed extract; kDa, kilodalton.

Figure 4. Antigen accumulation in transgenic seeds as determined by ELISA and given as percentage of TSP.

Values are the mean of three biological repeats, error bars represent the standard deviation. Accumulation of the GP3, GP4 and GP5 antigens is depicted as white, grey and black bars, respectively. Abbreviation used: TSP, total soluble protein.

Figure 5. IMAC purification of selected antigens.

One gram of homozygous seed stocks containing 0.14-Tm or 7.37 mg of GFP:GP4-Tm was extracted and subjected to IMAC. Purification of the GP4-Tm was analyzed by 12% SDS-PAGE and Coomassie Blue staining (A) or western blotting (B). Purification of the GFP:GP4-Tm was analyzed by SDS-PAGE (15%) and Coomassie Blue staining (C) or western blotting (D). All gels were run under reducing conditions and an antibody against the His₆-tag was used to detect the antigens during western blot analysis. Abbreviations used: S, sample; F, flow through; W, wash; E, elution; kDa, kilodalton.

Figure 6. Protein-A affinity chromatography of selected antigens.

For each antigen, one gram of homozygous seeds (equivalent to 0.15 mg GP4-Tm:mFc3, 2.06 mg GP4-Tm:pFc and 5.15 mg GP4-Tm:mFc2a) was extracted and subjected to protein-A chromatography. Purification of GP4-Tm:pFc (A), GP4-Tm:mFc2a (B) and GP4-Tm:mFc3 (C) was analyzed by 12% SDS-PAGE and Coomassie Blue staining (upper panels) or western blotting (lower panels). All gels were run under reducing conditions and the α-GP4 mAb XVI 11C was used to detect the antigens during western blotting. Abbreviations used: S, sample; F, flow through; W, wash; E, elution; kDa, kilodalton.

Figure 7. Characterization of the immunogenicity of selected GP4 antigens in a mouse model.

Per antigen, eight mice were vaccinated twice with 5 µg antigen mixed with an oil-in-water adjuvant. The booster vaccination was administered at week three (both vaccinations are depicted as black arrows). (A) Antigen-specific IgG titers induced by the GFP:GP4-Tm, GP4-Tm:mFc2a and sGP4-Tm:mFc2a at weeks two, four, and six after vaccination as measured by ELISA. Per vaccinated group, the individual values for each mouse and the mean (horizontal line) are given. (B) The titers of the sera collected at week six when tested via IPMA. Per vaccinated group the number of mice with a given titer is indicated. (C) The sera at week six of the GP4-Tm:mFc2a vaccinated mice were subjected to a seroneutralization assay to determine their virus neutralizing activity. The mean percentage of virus reduction (n =  eight mice) in function of the dilution is given, error bars represent the standard deviation.

Figure 8. Characterization of the immunogenicity of selected antigens carrying the pFc in a porcine model.

Three piglets were vaccinated twice with an antigen cocktail consisting of purified GP3-Tm:pFc, GP4-Tm:pFc and GP5-Tm:pFc mixed with an oil-in-water adjuvant. The booster vaccination was administered at week four (both vaccinations are depicted as black arrows). At different time points, the titers of IgG antibodies specific for GP3-Tm (A), GP4-Tm (B) and GP5-Tm (C) was determined. The collected sera were also tested via IPMA and titers of sera collected from week zero to six given (D).