Mucosal passive immunization is the transfer of active antibodies from one organism
to the mucosal surfaces of another organism for preventing or treating infectious diseases.
Mucosal passive immunization has a great potential for the prevention and treatment of
enteric infections like Rotavirus, which causes more than 114 million episodes of diarrhoea
annually with a death toll of more than 450.000 per year. However, the high cost of
recombinant antibodies with the current manufacturing systems based on mammalian cells
hampers the production of the high antibody quantities required for passive immunization
strategies. Alternative expression platforms such as plants could provide higher scalability and
reduced costs. Moreover, the use of edible plant organs, which are Generally¿Regarded¿As¿
Safe (GRAS), could reduce manufacturing costs even further by easing the requirements for
antibody purification. We analyze here the feasibility of utilizing fruits as inexpensive
biofactories of human antibodies that can be orally delivered as crude extracts or partially
purified formulations in mucosal passive immunization strategies.
In the first section of this thesis, the construction of tomato plants producing a model
human Immunoglobulin A (IgA) against rotavirus in their fruits is described. As a result, an elite
homozygous line was obtained whose fruits produced on average 41 ¿g of IgA per gram of
fresh weigh, equivalent to 0.69 mg IgA per gram of dry tomato powder. Minimally processed
products derived from IgA¿expressing tomatoes were shown to strongly inhibit virus infection
in an in vitro neutralization assay. Moreover, in order to make IgA¿expressing tomatoes easily
distinguishable from wild¿type tomatoes, they were sexually crossed with a transgenic tomato
line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors,
which confer purple colour to the fruit. The resulting transgenically¿labelled purple tomatoes
contained not only high levels of recombinant neutralizing human IgA but also increased
amounts of anthocyanins.
In the second section of the thesis the composition of IgA¿expressing tomatoes was
analyzed in search of possible unintended effects that could compromise the GRAS status of
the final product. To this end, transgenic IgA¿tomatoes were compared with wild type
tomatoes and also commercial tomato varieties using proteomic and metabolomic
approaches. 2D¿DIGE gels coupled with LC¿MSMS for protein identification showed that all the
uptrend differential proteins detected corresponded only to immunoglobulin chains or
antibody fragments. On the other hand, non¿targeted metabolite data obtained by UPLC¿MS / Juárez Ortega, P. (2014). Production of recombinant Immunoglobulin A in plants for passive immunotherapy [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/37015 / TESIS
| Identifer | oai:union.ndltd.org:upv.es/oai:riunet.upv.es:10251/37015 |
| Date | 14 April 2014 |
| Creators | Juárez Ortega, Paloma |
| Contributors | Granell Richart, Antonio, Orzáez Calatayud, Diego Vicente, Universitat Politècnica de València. Departamento de Biotecnología - Departament de Biotecnologia |
| Publisher | Universitat Politècnica de València |
| Source Sets | Universitat Politècnica de València |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/acceptedVersion |
| Source | Riunet |
| Rights | http://rightsstatements.org/vocab/InC/1.0/, info:eu-repo/semantics/openAccess |
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