Oral immunisation treatments and number of sheep assigned to each group.

<p>Oral immunisation treatments and number of sheep assigned to each group.</p

LTB-specific IgG (A) and IgA (B) antibody titres in abomasum mucus following oral immunisation with four doses of control or LTB-transgenic plant materials.

<p>The horizontal lines represent geometric means. Black symbols denote positive responders defined as sheep with antibody titres at least three standard deviations above the control mean, non-responders are indicated by grey symbols.</p

Relative abundance of LTB-specific IgG (A) and IgA (B) at different sections of the sheep small intestine following oral immunisation with four doses of control or LTB-transgenic plant materials.

<p>The horizontal lines represent geometric means. Black symbols denote positive responders defined as sheep with antibody titres at least three standard deviations above the control mean, non-responders are indicated by grey symbols.</p

LTB-specific IgG antibody titres in serum collected from sheep before immunisation with LTB-Leaf (A), LTB-HR (B) or control vaccines (C).

<p>Black symbols denote positive responders defined as sheep with antibody titres at least three standard deviations above the control mean, non-responders are indicated by grey symbols. The horizontal lines represent geometric means, statistical analysis (Student's t-test determined a significant difference between the means of the control and LTB-Leaf groups after four doses, p&lt;0.05).</p

LTB-specific antibody titres detected in intestinal washes performed at four sites along the first 11

<p> <b>m of the sheep small intestine following oral immunisation with four doses of LTB-Leaf (A and D, IgG and IgA respectively), LTB-HR (B and E, IgG and IgA respectively) or control (C and F, IgG and IgA respectively).</b> Sections 1, 2, 3 and 4 are defined as the first 0–0.5 m, 3.5–4 m, 7–7.5 m and 10.5–11 m respectively from the abomasum/duodenum junction. Black symbols denote positive responders defined as sheep with antibody titres at least three standard deviations above the control mean, non-responders are indicated by grey symbols.</p

Summary of the different experimental groups and candidate vaccine formulations.

<p>All HA antigen treatments consisted of 5 µg of HA. 20% alum (aluminium hydroxide) was added as an adjuvant where indicated.</p>*<p>Lower case “c” refers to the control group for each designated HA treatment group.</p

Characterisation of <i>N. tabacum X N. glauca</i> hybrids.

<p>3a. Floral and vegetative phenotypes of the hybrid (centre) compared with parentals. All plants in the bottom panel are eight weeks old. 3b. Molecular evidence for the presence of both parental genomes in <i>N. tabacum X N. glauca</i> interspecific hybrids. PCR was used to detect species-specific length variability in intron 5 of <i>Nicotiana QPT</i> paralogues. <i>N. tabacum</i> LAFC 53 generates different sized bands from <i>N. glauca.</i> Parental DNA refers to genomic <i>N. glauca</i> and <i>N. tabacum</i> LAFC 53 mixed <i>in vitro.</i> DNA-free refers to PCR performed without template. 3c. Microscopic analysis of pollen. Panels i–iii show pollen after initial staining with acetocarmine. Panels iv–vi shows pollen after incubation in pollen germination medium. Flanking panels show pollen from parental species as indicated. Centre panel shows non-viable hybrid pollen. All scale bars represent 50 µm. 3d. ELISA analysis of HA levels in individual interspecific hybrid plants. Data represents means of triplicate analysis ± SEM. DW = dry weight. All lines are derived from T_o parental <i>Nt</i> LAFC-HA pDAB4493-8.</p

Analysis of interspecific hybrids for alkaloid content.

<p>4a. Chromatograms of the pyridine alkaloid profile in the parental and hybrids lines after removal of plant apices. (i) Nicotinic acid mononucletide was extracted as a background metabolite in the methanol-soluble fraction, (ii) Anabasine profile, (iii) Nicotine profile. 4b. Total pyridine alkaloid levels in vegetative regenerating shoots of parental and WT hybrid controls and HA-containing interspecific hybrid plants. Analysis was undertaken one week after removal of plant apices; graphs represent the mean of three separate plants per treatment (± SEM), ND = None Detected. DW = dry weight.</p

Capacity of HA-containing plant extracts to elicit an immune response.

<p>5a. HA-specific systemic immune response of mice. The horizontal lines represents the geometrical means of HA specific IgG titres at day 28, while the data points represent IgG titres from individual mice. Detailed description of treatments is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035688#pone-0035688-t001" target="_blank">Table 1</a>. Solid dots indicate response to samples containing HA antigen. Open circles indicate responses to samples without HA antigen. A significant difference exits between HA positive samples and control samples (p = 0.0002). 5b. Detection of anti-HA specific IgG isotypes in mice sera on day 28. The horizontal lines represent the geometrical means of IgG isotype titres, while the dots represent IgG isotype titres from individual mice. Detailed description of treatments is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035688#pone-0035688-t001" target="_blank">Table 1</a>.</p

Analysis of T₀<i>N. tabacum</i> LAFC 53 plant lines transformed with HA constructs.

<p>2a. The mean HA content in <i>Nt</i> LAFC-HA plants transgenic for each construct type quantified using ELISA analysis. *Mean levels of HA in plants transgenic for pDAB4493 were significantly different (p&lt;0.05; t-test) to those containing pDAB4492. Nt = non-transgenic <i>N. tabacum</i> var. LAFC 53. Error bars represent standard error of mean (SEM) (n = 40 for plants transgenic for pCHA; n = 45 for plants transgenic for pDAB4492; n = 52 for plants transgenic for pDAB4493), FW = fresh weight. 2b. Western analysis of leaf extracts from representative <i>Nt</i> LAFC-HA plants. Extracts from plants containing HA constructs were loaded in central lanes as indicated. Purified HA was loaded as a control in the left lane whilst leaf extract from non-transgenic <i>N. tabacum</i> var. LAFC 53 was loaded in the right lane. All lanes contained 10 µg total soluble protein (TSP). 2c. Detection of HA transgene in selected elite transgenic lines <i>of N. tabacum</i> containing the pDAB4493-HA construct. Southern blot hybridisation of <i>Hind</i> III digested genomic DNA isolated from Nt LAFC-HA transgenic plants probed with the HA gene.</p