An interspecific Nicotiana hybrid as a useful and cost-effective platform for production of animal vaccines

Abstract

The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs.

Figure 1. Constructs used to drive expression of HA gene in N. tabacum LAFC 53.

CsVMV = the cassava vein mosaic virus promoter. 4OCS-ΔMas = a chimeric synthetic promoter. Native HA = the coding region of haemagglutinin from the turkey Wisconsin HA5 AIV strain. Plant-codon optimised HA = the modified haemagglutinin coding sequence based on plant codon usage frequency. LB and RB represent the left and right T-DNA borders respectively. VSP 3′ = the terminator of the soybean vegetative storage protein. PAT = the phosphinothricin acetyl transferase gene.

Figure 2. Analysis of T₀ N. tabacum LAFC 53 plant lines transformed with HA constructs.

2a. The mean HA content in Nt 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<0.05; t-test) to those containing pDAB4492. Nt = non-transgenic N. tabacum 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 Nt 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 N. tabacum 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 of N. tabacum containing the pDAB4493-HA construct. Southern blot hybridisation of Hind III digested genomic DNA isolated from Nt LAFC-HA transgenic plants probed with the HA gene.

Figure 3. Characterisation of N. tabacum X N. glauca hybrids.

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 N. tabacum X N. glauca interspecific hybrids. PCR was used to detect species-specific length variability in intron 5 of Nicotiana QPT paralogues. N. tabacum LAFC 53 generates different sized bands from N. glauca. Parental DNA refers to genomic N. glauca and N. tabacum LAFC 53 mixed in vitro. 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 Nt LAFC-HA pDAB4493-8.

Figure 4. Analysis of interspecific hybrids for alkaloid content.

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.

Figure 5. Capacity of HA-containing plant extracts to elicit an immune response.

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 Table 1. 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 Table 1.

Figure 6. Haemagglutination inhibition (HI) titres of sera from immunized mice.

The geometrical means of the HI titres are expressed as reciprocals of the highest dilution of serum that inhibited four haemagglutinin units of virus. Values above the broken line (dilutions of ≥1∶40) indicates the presence of protective HI titres. A significant difference exits between HA samples and the non immunised control (p<0.0001).

Figure 7. Innate immune response in human peripheral blood mononuclear cells.

Cytokine production was measured by specific ELISA, absorbance 750 nm. Data from biological triplicates representative from two independent experiments involving two different blood donors. IFN, interferon; TNF, tumor necrosis factor. Gardiquimod and CL75 were used as controls (see methods).