Feasibilty of oral immunisation with LTB-based edible vaccines

This thesis describes the research to explore the feasibility of plants for oral vaccination. The research focussed on a model of LTB produced in potato tubers or ovalbumin (OVA) as antigen and tested in mice. A general introduction into the backgrounds of oral immunisation is given in Chapter 1. The next two chapters describe the optimisation of an immunisation protocol for edible vaccines by addressing the following questions: can the immune response be increased by more frequent doses of edible vaccines without negative effects on general health (Chapter 2)?; and can the immune status of the host be modified to react more efficiently to a subsequent oral boost (Chapter 3)?. Ovalbumin (OVA) was used as model antigen and administered via intragastric (IG) gavage to mice. The results of these studies led to a refined immunisation protocol in which one single subcutaneous, adjuvanted priming is followed three weeks later by oral boost immunisations on three alternating days (also referred to as the systemic prime/oral boost protocol). As a model for edible vaccines, the heat-labile enterotoxin subunit-B (LTB) of Escherichia coli was produced in potato tubers. This edible vaccine was either fed or administered orally to mice. Using the optimised immunisation protocol, local and systemic responses against LTB were induced (Chapter 4). Subsequently, the use of LTB as adjuvant for co-expressed antigens in edible vaccines was explored. A glycoprotein (E2) of the classical swine fever virus was co-expressed as fusion protein to LTB or expressed together with LTB in potatoes, resulting in E2-LTB and E2 + LTB potatoes, respectively. LTB fused to these antigens retained its biological activity (GM1-binding). The expression levels of LTB varied from 0.25% in LTB-transgenic plants to 0.01% LTB per total soluble protein in E2-LTB-transgenic plants. The levels of LTB as fusion-proteins were lower than those of LTB alone or co-expressed with E2. LTB, E2 + LTB, and CVP-LTB producing tubers were immunogenic upon subcutaneous immunisation and significant antibody responses against LTB were detected. The response towards the co-expressed antigens were low or undetectable. Probably, the antigen dose of the co-expressed antigen was too low and the adjuvant capacity of LTB was insufficient (Chapter 5). Further research is required to improve the carrier and adjuvant function of LTB. Another point of concern is that besides adjuvant activity, LT,CT and their B-subunits are also known to be capable to induce oral tolerance. Future research must concentrate on more effective carrier-molecules or adjuvants devoid of tolerating properties. The chapters 4 and 6 clearly demonstrated that IG gavage of tuber or chow extracts induced higher antibody responses than similar doses of antigen taken up with feed or drinking water. It was concluded that the route of oral administration is at least as important than the vaccine composition. The difference between feeding and oral administration must be taken into account when the feasibility of edible vaccines is assessed (Chapter 6). In Chapter 7, the general findings of this thesis are discussed. The proposed systemic prime/triple dose oral boost protocol appeared to be applicable for oral administration and oral intake of edible vaccines in particular. However, significant adverse effects were observed after tuber intake. The ideal edible vaccine in plants has sufficient levels of antigen, is easy to propagate under a wide range of conditions and is not toxic when given the amounts required. In this respect, the use of potatoes has several drawbacks. Consumption of raw potatoes is not preferable and cooking might denature the antigen. For future edible vaccine studies, a more suitable plant with relatively high expression levels should be chosen (e.g. tomato)

Enhancement of protective immune responses by oral vaccination with Saccharomyces cerevisiae expressing recombinant Actinobacillus pleuropneumoniae ApxIA or ApxIIA in mice

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1β and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-α increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection

Comparison of immune responses in parenteral FaeG DNA primed pigs boosted orally with F4 protein or reimmunized with the DNA vaccine

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Transgenic tomatoes expressing human beta-amyloid for use as a vaccine against Alzheimer’s disease

Human β-amyloid (Aβ) is believed to be one of the main components of Alzheimer’s disease, so reduction of Aβ is considered a key therapeutic target. Using Agrobacterium-mediated nuclear transformation, we generated transgenic tomatoes for Aβ with tandem repeats. Integration of the human Aβ gene into the tomato genome and its transcription were detected by PCR and Northern blot, respectively. Expression of the Aβ protein was confirmed by western blot and ELISA, and then the transgenic tomato line expressing the highest protein level was selected for vaccination. Mice immunized orally with total soluble extracts from the transgenic tomato plants elicited an immune response after receiving a booster. The results indicate that tomato plants may provide a useful system for the production of human Aβ antigen

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The objective of this research is to analyze, from a bioethical and legal perspective, one of the particularly complex situations in which a woman -and her family- might find themselves in front of a diagnosis of anencephalic fetus, and to consider weather the decision of interrupting that pregnancy would or not be ethically justifiable.El objetivo de este trabajo es abordar, desde la perspectiva bioética y jurídica una de las situaciones particularmente complejas, en las que puede encontrarse una mujer -y su grupo familiar- frente al diagnóstico de la gestación de un feto anencefálico, y analizar si la decisión de interrumpir el embarazo puede o no resultar éticamente justificable.L'objectif du travail est d'aborder, du point de vue bioéthique et juridique, une des situations particulièrement complexes dans laquelle peut se trouver une femme -et son groupe familial- face au diagnostic d'un foetus anencéphalique, et analiser la situation pour voir si la décision d'interrompre la grossesse pourrait ou non être éthiquement justifiable.O objetivo deste trabalho é analisar, desde uma perspectiva bioética e jurídica uma da situações particularmente complexas, em que pode encontrar-se uma mulher -e seu grupo familiar- frente ao diagnóstico de gestação de feto anencefálico. Pretende considerar se a decisão de interrupção da gestação poderá ou não ter justificação ética

Improvement of the systemic prime/oral boost strategy for systemic and local responses.

This paper describes oral boost immunisations of primed animals as an alternative oral vaccination strategy. Mice were primed orally (PO), intranasally (IN), subcutaneously (SC), or intraperitoneally (IP) with ovalbumin (OVA) with or without adjuvant. Boost immunisations were given orally with or without cholera toxin (CT) as adjuvant. Prime immunisations induced variable IgA and IgG1 titres in serum depending on the route. A subsequent oral boost increased these titres. Use of an adjuvant in the priming significantly increased serum IgA and, to a lesser extend, IgG1. Oral boost immunisation induced significantly higher serum IgA titres in animals primed via the SC, IP and the IN route compared to the PO route. This was independent of the use of CT. Three oral boosts with OVA plus 5 ¿g CT given in 5 days to primed mice revealed higher IgA titres compared to single oral boosts and anti-OVA IgA titres in faeces were also detected. Finally, we put together our findings and propose a systemic priming/oral boost strategy in which mice were primed via the SC route with 100 ¿g OVA plus 50 ¿g Butyl16-p(AA), and subsequently orally boosted with three doses of 300 ¿g OVA plus 5 ¿g CT each. We concluded that oral immunisation is more effective in IN, SC, or IP primed mice than in PO primed mice, and that the IgA antibody response in serum and faeces can be improved by increasing the immunisation frequency and the use of appropriate adjuvants in primary and boost immunisation. The here-formulated strategy improves the probability of success of oral vaccination. The results are discussed in the light of the development of edible vaccines