An Interspecific Nicotiana Hybrid as a Useful and Cost-Effective Platform for Production of Animal Vaccines

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

Transient Expression of Hemagglutinin Antigen from Low Pathogenic Avian Influenza A (H7N7) in Nicotiana benthamiana

The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species

EFSA Panel on Biological Hazards (BIOHAZ); Scientific Opinion on public health risks represented by certain composite products containing food of animal origin

This Opinion reviews the factors that affect microbial survival and growth in composite products, and in foods in general. It concludes that the main factors to be considered are: water activity, pH, temperature and duration of storage, processing, and intensity and duration of other non-thermal physical processes applied. Prevalence and concentration of the pathogens in food are important to determine the risk for consumers. The opinion presents a review of the quantitative microbiology models and databases that can be used to provide quantitative estimations of the impact of the above factors on the survival and growth of the main bacterial pathogens. In composite products, migration and diffusion of moisture and substances among the ingredients may change their physico-chemical parameters, particularly at the interfaces. Therefore, the assessment of the risk posed by composite products needs to consider the combinations of parameters most permissive to survival and growth of pathogens. Two complementary approaches are proposed for the identification and profiling of microbiological hazards in different specific composite products. The first one is based on past outbreaks and prevalence of hazards in the products and leads to the conclusion that the most frequent hazard-composite product combinations are Salmonella in cakes and bakery products. The second one consists in decision tools based on the impact on the pathogens of food composition and food processing. Categorisation of the risk for composite products requires information on their composition, processing and further handling, which can largely differ for foods belonging to the same category. Further conditions may influence the risk and should be verified, i.e. hygienic conditions during preparation of the composite products and their ingredients, shelf-life conditions, and reliability of cooking by consumers to inactivate pathogens. The decision tools developed apply to all composite products considered by the mandate, as well as to all other foods. © European Food Safety Authority, 201

Current Status and Perspectives of the Molecular Farming Landscape

This chapter provides an overview of the current state of molecular farming, which is characterized by the development of sizeable production capacities, a greater focus on downstream processing and scale-up, and the targeting of niche products that take advantage of favorable or even unique features of the plant production host. This situation reflects the maturation of plant-based production platforms and a drive toward commercialization with a range of serious product candidates in both the pharmaceutical and non-pharmaceutical field