Evaluation of Unintended Effects in the Composition of Tomatoes Expressing a Human Immunoglobulin A against Rotavirus Paloma Juarez, Asun Fernandez-del-Carmen, Jose L. Rambla, Silvia Presa, Amparo Mico, Antonio Granell, and Diego Orzaez* Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientı ́ ficas, Universidad Polite ́ cnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain * S Supporting Information ABSTRACT: 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. KEYWORDS: tomato, IgA, antibody, rotavirus, unintended effects, metabolomics, proteomics ■ INTRODUCTION Antibody-based treatments for mucosal passive immunization (MPI) have great potential in human and veterinary medicine for the prevention of infection diseases. MPI is particularly advantageous for the treatment of enteric diseases. 1-3 Generally, passive antibody-based treatments require large amounts of antibodies to be delivered to the target mucosa to ensure protection. 4 The high specificity of recombinant monoclonal antibodies makes them excellent candidates for MPI; however, the increasing costs of producing recombinant antibodies in the preferred mammalian cell platforms has hampered their use in MPI. As an alternative, neutralizing recombinant antibodies aimed at MPI can be inexpensively produced in edible plant organs. Many seeds, fruits, leaves, tubers, and roots are considered safe and palatable for human consumption in minimally processed formulations without heat treatment; 5 therefore, it has been proposed that antibodies produced in plant organs with generally regarded as safe (GRAS) status could be delivered as dose-controlled ingredients in partially processed formula- tions without the need for exhaustive purification. 4 This would certainly reduce the manufacturing costs, as plant platforms are reportedly easier to scale up than other platforms, for example, those based on fermentation. 5 The lack of exhaustive purification can, on the other hand, become a drawback when the composition of the final product ultimately results from an event of genetic modification. It could be argued that, besides producing a recombinant antibody, transgenesis could eventually lead to unintended effects in the final fruit composition. 6,7 Such unintended effects could be derived from the integration of the transgene, from biological interactions caused by transgene-encoding proteins, or from spurious somaclonal mutations. Unintended effects have become one of the most controversial issues in biological safety debates. 8 Despite the evidence accumulated during the past 20 years indicating that transgenesis could be even less disruptive of food composition than traditional breeding, 8-10 the absence of unintended, deleterious effects in the composition of genetically modified edible plants organs expressing recombinant antibodies has never been assessed. Recently, our group reported a model human immunoglo- bulin A (IgA) for passive protection against the enteric pathogen rotavirus, produced in transgenic tomato fruits. Dry formulations suitable for oral intake and compatible with long- term storage presented a high concentration of active antibodies (≈0.7 mg/g DW), which were shown to inhibit virus infections in an in vitro virus neutralization assay. Thus, minimally processed fruit-derived formulations containing recombinant IgA were proposed as potential vehicles for low- cost MPI treatments. 11 The availability of IgA-producing fruits provides an opportunity to test for the possible unintended effects that the expression of a human antibody could have on the final composition of the fruit and to infer the possible deleterious effects on human health that such unintended effects could have. Safety concerns in tomato fruit composition arise funda- mentally from two sources: proteins (allergens) and secondary metabolites (toxicants). So far, only three tomato protein allergens, Lyc e 1 (profilin), Lyc e 2 (invertase), and Lyc e 3 Received: May 16, 2014 Revised: July 18, 2014 Accepted: July 27, 2014 Published: July 28, 2014 Article pubs.acs.org/JAFC © 2014 American Chemical Society 8158 dx.doi.org/10.1021/jf502292g | J. Agric. Food Chem. 2014, 62, 8158-8168