Production of recombinant Immunoglobulin A in plants for passive immunotherapy

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¿MSJuárez 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/10251/37015TESI

Analyzing adaptation to climate change in the water and the agricultural sectors in the Spanish Guadiana basin.

1. Introduction: setting and problem definition 2. The Adaptation Pathway –2.1 Stage 1: appraising risks and opportunities •Step 1: Impact analysis •Step 2: Policy analysis •Step 3: Socio-institutional analysis –2.2 Stage 2: appraising and choosing adaptation opt ions •Step 4: identifying and prioritizing adaptation o ptions 3. Conclusion

Ahora / Ara

La cinquena edició del microrelatari per l’eradicació de la violència contra les dones de l’Institut Universitari d’Estudis Feministes i de Gènere «Purificación Escribano» de la Universitat Jaume I vol ser una declaració d’esperança. Aquest és el moment en el qual les dones (i els homes) hem de fer un pas endavant i eliminar la violència sistèmica contra les dones. Ara és el moment de denunciar el masclisme i els micromasclismes començant a construir una societat més igualitària. Cadascun dels relats del llibre és una denúncia i una declaració que ens encamina cap a un món millor

Neutralizing antibodies against rotavirus produced in identity¿preserved purple tomatoes

Edible fruits are inexpensive biofactories of recombinant molecules for oral intake, which can be then delivered as crude extracts or partially purified formulations. Our group aims at the production of human antibodies for passive protection against mucosal pathogens in identitypreserved fruits. Tomato plants were transformed with a recombinant human immunoglobulin A (IgA_2A1) selected against the VP8* peptide of rotavirus SA11 strain. The expression levels of IgA_2A1 reached up to 5% of the total soluble protein in the transgenic fruit. Tomato crude extracts showed specific anti-VP8* binding activity up to 1/192 (FreshWeight:volume) dilutions, confirming the efficient assembly of IgA molecules with specific antigen-binding activity. Moreover, transgenic tomato extracts showed neutralizing activity against rotavirus SA11 strain in an in vitro neutralization assay. In order to make IgA_2A1 tomatoes easily distinguishable from the wild type, IgA_2A1 lines were crossed with metabolically¿engineered purple tomatoes expressing Anthirrhinum majus Rosea1 and Delila transcription factors. As a result, identity-preserved tomato fruits were obtained whose purple¿coloured juice contained high levels of health-promoting anthocyanins in combination with neutralizing anti¿rotavirus human IgA.Juárez Ortega, P. (2010). Neutralizing antibodies against rotavirus produced in identity¿preserved purple tomatoes. http://hdl.handle.net/10251/13643Archivo delegad

Recombinant jacalin-like plant lectins are produced at high levels in Nicotiana benthamiana and retain agglutination activity and sugar specificity

The plant kingdom is an underexplored source of valuable proteins which, like plant lectins, display unique interacting specificities. Furthermore, plant protein diversity remains under-exploited due to the low availability and heterogeneity of native sources. All these hurdles could be overcome with recombinant production. A narrow phylogenetic gap between the native source and the recombinant platform is likely to facilitate proper protein processing and stability; therefore, the plant cell chassis should be specially suited for the recombinant production of many plant native proteins. This is illustrated herein with the recombinant production of two representatives of the plant jacalin-related lectin (JRLs) protein family in Nicotiana benthamiana using state-of-the-art magnICON technology. Mannose-specific Banlec JRL was produced at very high levels in leaves, reaching 1.0 mg of purified protein per gram of fresh weight and showing strong agglutination activity. Galactose-specific jacalin JRL, with its complicated processing requirements, was also successfully produced in N. benthamiana at levels of 0.25 mg of purified protein per gram of fresh weight. Recombinant Jacalin (rJacalin) proved efficient in the purification of human IgA1, and was able to discriminate between plant-made and native IgA1 due to their differential glycosylation status. Together, these results show that the plant cell factory should be considered a primary option in the recombinant production of valuable plant proteins. (C) 2012 Elsevier B.V. All rights reserved.We wish to acknowledge Prof. Gleba and Dr. Klimyuk from ICON Genetics for providing the magnICON expression system. We also thank Prof. R.P. Sharma, University of Hyderabad, India, for kindly providing us with jackfruit seeds and Dr. Luis Rodenas at the UPV Animal Sciences Department for his help with samples for the agglutination analysis. This work has been supported by Grant BIO2010-15384 from MINECO, Spain, and P. Juarez FPU fellowship AP2008-01140.Fernández Del Carmen, MA.; Juárez Ortega, P.; Presa Castro, S.; Granell Richart, A.; Orzáez Calatayud, DV. (2013). Recombinant jacalin-like plant lectins are produced at high levels in Nicotiana benthamiana and retain agglutination activity and sugar specificity. Journal of Biotechnology. 163(4):391-400. https://doi.org/10.1016/j.jbiotec.2012.11.017S391400163

Evaluation of unintended effects in the composition of tomatoes expressing a human immunoglobulin A against Rotavirus

The production of neutralizing immunoglobulin A (IgA) in edible fruits as a means of oral passive immunization is a promising strategy for the inexpensive treatment of mucosal diseases. This approach is based on the assumption that the edible status remains unaltered in the immunoglobulin-expressing fruit, and therefore extensive purification is not required for mucosal delivery. However, unintended effects associated with IgA expression such as toxic secondary metabolites and protein allergens cannot be dismissed a priori and need to be investigated. This paper describes a collection of independent transgenic tomato lines expressing a neutralizing human IgA against rotavirus, a mucosal pathogen producing severe diarrhea episodes. This collection was used to evaluate possible unintended effects associated with recombinant IgA expression. A comparative analysis of protein and secondary metabolite profiles using wild type lines and other commercial varieties failed to find unsafe features significantly associated with IgA expression. Preliminary, the data indicate that formulations derived from IgA tomatoes are as safe for consumption as equivalent formulations derived from wild type tomatoes.This work has been funded by Grant BIO2010-15384 from Plan Nacional I + D of the Spanish Ministry of Science. P.Juarez is the recipient of a FPU fellowship.Juárez Ortega, P.; Fernández Del Carmen, MA.; Rambla Nebot, JL.; Presa Castro, S.; Mico, A.; Granell Richart, A.; Orzáez Calatayud, DV. (2014). Evaluation of unintended effects in the composition of tomatoes expressing a human immunoglobulin A against Rotavirus. Journal of Agricultural and Food Chemistry. 62(32):8158-8168. https://doi.org/10.1021/jf502292gS81588168623