Production of a subunit vaccine candidate against porcine post-weaning diarrhea in high-biomass transplastomic tobacco

Post-weaning diarrhea (PWD) in piglets is a major problem in piggeries worldwide and results in severe economic losses. Infection with Enterotoxigenic Escherichia coli (ETEC) is the key culprit for the PWD disease. F4 fimbriae of ETEC are highly stable proteinaceous polymers, mainly composed of the major structural subunit FaeG, with a capacity to evoke mucosal immune responses, thus demonstrating a potential to act as an oral vaccine against ETEC-induced porcine PWD. In this study we used a transplastomic approach in tobacco to produce a recombinant variant of the FaeG protein, rFaeG(ntd/dsc), engineered for expression as a stable monomer by N-terminal deletion and donor strand-complementation (ntd/dsc). The generated transplastomic tobacco plants accumulated up to 2.0 g rFaeG(ntd/dsc) per 1 kg fresh leaf tissue (more than 1% of dry leaf tissue) and showed normal phenotype indistinguishable from wild type untransformed plants. We determined that chloroplast-produced rFaeG(ntd/dsc) protein retained the key properties of an oral vaccine, i.e. binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. Additionally, the plant biomass matrix was shown to delay degradation of the chloroplast-produced rFaeG(ntd/dsc) in gastrointestinal conditions, demonstrating a potential to function as a shelter-vehicle for vaccine delivery. These results suggest that transplastomic plants expressing the rFaeG(ntd/dsc) protein could be used for production and, possibly, delivery of an oral vaccine against porcine F4+ ETEC infections. Our findings therefore present a feasible approach for developing an oral vaccination strategy against porcine PWD

Bringing plant-based veterinary vaccines to market: Managing regulatory and commercial hurdles

AbstractThe production of recombinant vaccines in plants may help to reduce the burden of veterinary diseases, which cause major economic losses and in some cases can affect human health. While there is abundant research in this area, a knowledge gap exists between the ability to create and evaluate plant-based products in the laboratory, and the ability to take these products on a path to commercialization. The current report, arising from a workshop sponsored by an Organisation for Economic Co-operation and Development (OECD) Co-operative Research Programme, addresses this gap by providing guidance in planning for the commercialization of plant-made vaccines for animal use. It includes relevant information on developing business plans, assessing market opportunities, manufacturing scale-up, financing, protecting and using intellectual property, and regulatory approval with a focus on Canadian regulations

A Plant-produced candidate subunit vaccine reduces shedding of Enterohemorrhagic <em>Escherichia col</em>i in ruminants

Enterohemorrhagic Escherichia coli (EHEC) are commonly present in the gastrointestinal tract of cattle and cause serious infectious disease in humans. Immunizing cattle against EHEC is a promising strategy to decrease the risk of food contamination; however, veterinary vaccines against EHEC such as Econiche have not been widely adopted by the agricultural industry, and have been discontinued, prompting the need for more cost-effective EHEC vaccines. The objective of this project is to develop a platform to produce plant-made antigens for oral vaccination of ruminants against EHEC. Five recombinant proteins were designed as vaccine candidates and expressed transiently in Nicotiana benthamiana and transplastomically in Nicotiana tabacum. Three of these EHEC proteins, NleA, Stx2b, and a fusion of EspA accumulated when transiently expressed. Transient protein accumulation was the highest when EHEC proteins were fused to an elastin-like polypeptide (ELP) tag. In the transplastomic lines, EspA accumulated up to 479mgkg(-1) in lyophilized leaf material. Sheep that were administered leaf tissue containing recombinant EspA shed less E. coli O157:H7 when challenged, as compared to control animals. These results suggest that plant-made, transgenic EspA has the potential to reduce EHEC shedding in ruminants

Mucosal Immunization with Spore-Based Vaccines against Mannheimia haemolytica Enhances Antigen-Specific Immunity

Background: Mannheimia haemolytica is a bovine respiratory pathogen commonly associated with bacterial bronchopneumonia. Current vaccine strategies have shown variable efficacy in feedlot cattle, and therefore novel vaccines are needed. Bacillus subtilis spores have been investigated as a mucosal vaccine platform, due to their ability to bind and present antigens to the mucosa and act as an adjuvant. The aim of this study was to develop two spore-based mucosal vaccines targeting M. haemolytica and evaluate their immunogenicity in mice. Methods: Two antigen constructs composed of cholera toxin B subunit, M. haemolytica leukotoxin, and either the M. haemolytica outer membrane protein PlpE (MhCP1) or GS60 (MhCP2) were synthesized, purified and then bound to spores as vaccines. In two separate mice trials, the spore-bound vaccines (Spore-MhCP1 and Spore-MhCP2) were administered to mice through intranasal and intragastric routes, while free antigens were administered intranasally and intramuscularly. Unbound spores were also evaluated intranasally. Antigen-specific serum IgG and mucosal IgA from bronchoalveolar lavage, feces, and saliva were measured after vaccination. Mice sera from all treatment groups were assessed for their bactericidal activity against M. haemolytica. Results: In both mice experiments, intramuscular immunization induced the strongest serum IgG antibody response. However, the intranasal administration of Spore-MhCP1 and Spore-MhCP2 elicited the greatest secretory IgA-specific response against leukotoxin, PlpE, and GS60 in bronchoalveolar lavage, saliva, and feces (p < 0.05). Compared to the intranasal administration of free antigen, spore-bound antigen groups showed greater bactericidal activity against M. haemolytica (p < 0.05). Conclusions: Since intranasally delivered Spore-MhCP1 and Spore-MhCP2 elicited both systemic and mucosal immune responses in mice, these vaccines may have potential to mitigate lung infection in cattle by restricting M. haemolytica colonization and proliferation in the respiratory tract. The efficacy of these mucosal spore-based vaccines merits further assessment against M. haemolytica in cattle.Land and Food Systems, Faculty ofNon UBCReviewedFacultyResearche

Purification of rFaeG_ntd/dsc from crude plant extract and quantification.

<p>(a) rFaeG_ntd/dsc was extracted from 5 g of mature transplastomic leaf tissue and purified. The initial volume of the extract was 50 ml; 3 µl of the extract from each step of the procedure were resolved by SDS-PAGE and stained. Lane 1 - Initial extract from leaf tissue, pH = 7.5; lane 2 - extract acidified to pH = 2 and centrifuged; lane 3 - clarified extract neutralized to pH = 7.4; Lane 4 - flowthrough from IMAC column; Lane 5 - wash with 20 mM imidazole; Lane 6 - elution of purified rFaeG_ntd/dsc; Lane 7 - 0.5 µg of BSA as loading control; kDa - protein molecular weight marker. (b) Purified rFaeG_ntd/dsc was quantified using densitometry. Dilutions of the purified rFaeG_ntd/dsc protein (lanes 1 through 7) were resolved in SDS-PAGE gel along with known amounts of BSA (lanes 8–14; 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05 µg BSA, respectively) and stained. BSA bands were used for generation of a standard curve (<i>R²</i> = 0.987; <i>p</i> = 0.01) and extrapolating rFaeG_ntd/dsc concentration. kDa - molecular weight marker.</p

Accumulation of chloroplast-targeted, transiently-expressed rFaeG_ntd/dsc.

<p>Transient expression of the rFaeG_ntd/dsc protein via agroinfiltration in <i>Nicotiana benthamiana</i> leaves was examined by SDS-PAGE and staining (a), and immunoblot analysis (b). Lanes 1 and 2−5.0 µg of protein extract of leaves co-infiltrated with <i>Agrobacteria</i> carrying chloroplast-targeted rFaeG_ntd/dsc and the p19 viral suppressor of post-transcriptional gene silencing (1), or p19 alone as negative control (2). rFaeG_ntd/dsc is indicated with a black rhomb, higher bands likely correspond to rFaeG_ntd/dsc with partially cleaved transit peptide; Lane 3−0.5 µg purified F4_ad fimbriae as positive control, the F4 native FaeG is indicated with a black triangle; the ∼2 kDa difference in size of rFaeG_ntd/dsc (29 kDa) and the native FaeG (27 kDa) is due to the additional complementing fused domain.</p

Accumulation levels of rFaeG_ntd/dsc in transplastomic leaf tissue.

<p>(a) Samples of equal volume (4 µl) were prepared from crude extract fractions. Lane 1 - WT extract (negative control); lanes 2, 3 and 4 represent crude extract of 0.4 mg of leaf tissue, re-extracted pellet, and clarified extract, respectively, where clarified extract contains 5 µg TSP. The rFaeG_ntd/dsc yield was estimated using a standard curve (<i>R²</i> = 0.993) of known amounts of purified rFaeG_ntd/dsc (lanes 5 through 8∶2 µg, 1 µg, 0.5 µg and 0.25 µg, respectively). (b) No variation in rFaeG_ntd/dsc accumulation was observed in transplastomic clones (C1, C2) after dark (D) or after light (L) periods. Image is representative of sampling on three different days, 1 µg TSP was used per lane. WT =  untransformed control.</p

Homoplastomic lines show normal phenotype.

<p>(a) A schematic representation of the chloroplast transformation cassette (pCT-rFaeG_ntd/dsc). The cassette was designed to integrate between the <i>trnI (tRNA-Ile)</i> and <i>trnA (tRNA-Ala)</i> genes of the tobacco plastome. The wild type (WT) plastome <i>trnI - trnA</i> region is shown at the bottom. Expected sizes of <i>Rsr</i> II-digested fragments are indicated. Thick black lines represent hybridization sites for the probe used in Southern blot analyses. IEE  =  intercistronic expression element with the Shine-Dalgarno sequence from the 5′ UTR of bacteriophage T7 gene 10 fused to the 3′ end; aadA  =  gene encoding aminoglycoside 3′ adenylyltransferase for spectinomycin resistance; T<i>psbC</i>  = 3′ UTR of <i>psbC</i> from white poplar plastome; P<i>psbA</i>  = 5′ UTR and promoter of tobacco <i>psbA</i> gene. <i>rfaeG_ntd/dsc</i>  =  gene encoding the rFaeG_ntd/dsc protein variant. T<i>rbcL</i>  = 3′ UTR of <i>rbcL</i> from white poplar plastome. (b) Phenotypes of mature transplastomic tobacco cv. I 64 plants transformed with pCT-rFaeG_ntd/dsc (1 and 2) were indistinguishable from WT plants (3). A one-meter ruler was photographed to the left of each plant as size reference. (c) Confirmation of homoplastomy. Southern blot analysis of total plant DNA from 2 independent transformants and 1 untransformed plant displayed a single band of the expected size.</p

Chloroplast-produced rFaeG_ntd/dsc protein is recognized in F4 fimbriae-specific ELISA, partially polymerizes and specifically binds to the brush border of F4R+ small intestinal villi.

<p>(a) Both rFaeG_ntd/dsc and F4 fimbriae are recognized by a monoclonal anti-F4_ad fimbriae antibody in ELISA. (b) Purified F4 fimbriae (lane 1) and purified rFaeG_ntd/dsc (lane 2) were resolved under non-reducing conditions to assess polymerization. The F4 fimbriae sample displayed the formation of native FaeG polymers, number of subunits is indicated by stacked black triangles next to each band. Most of the rFaeG_ntd/dsc is present as monomers (denoted by black rhomb); formation of rFaeG_ntd/dsc dimers and trimers was also observed (two and three stacked black rhombs). (c) Adhesion of the rFaeG_ntd/dsc protein to the brush border of F4R+ small intestinal villi. Binding to the F4-specific receptors present on the apical surface of the epithelial cells, which line the brush border of F4R+ small intestinal villi is shown as a bright line on the edge of the sample, the result of excited FITC fluorochrome (indicated with white arrows, lower panel). rFaeG_ntd/dsc fails to bind to brush border of F4R− small intestinal villi. Images are representative of rFaeG_ntd/dsc adhesion to isolated villi of three F4R+ and two F4R− piglets. Bar: 50 µm.</p