There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β‐glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide‐spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse–human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv‐Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β‐glucan‐binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3‐linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv‐Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant‐derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.

Background: Infections caused by fungi are often refractory to conventional therapies and urgently require the development of novel options, such as immunotherapy. To produce therapeutic antibodies, a plant-based expression platform is an attractive biotechnological strategy compared to mammalian cell cultures. In addition to whole plants, hairy roots (HR) cultures can be used, representing an expression system easy to build up, with indefinite growth while handled under containment conditions. Results: In this study the production in HR of a recombinant antibody, proved to be a good candidate for human immunotherapy against fungal infections, is reported. Expression and secretion of this antibody, in an engineered single chain (scFvFc) format, by HR from Nicotiana benthamiana and Solanum lycopersicum have been evaluated with the aim of directly using the deriving extract or culture medium against pathogenic fungi. Although both Solanaceae HR showed good expression levels (up to 68 mg/kg), an optimization of rhizosecretion was only obtained for N. benthamiana HR. A preliminary assessment to explain this result highlighted the fact that not only the presence of proteases, but also the chemical characteristics of the growth medium, can influence antibody yield, with implications on recombinant protein production in HR. Finally, the antifungal activity of scFvFc 2G8 antibody produced in N. benthamiana HR was evaluated in Candida albicans growth inhibition assays, evidencing encouraging results. Conclusions: Production of this anti-fungal antibody in HR of N. benthamiana and S. lycopersicum elucidated factors affecting pharming in this system and allowed to obtain promising ready-to-use immunotherapeutics against C. albicans.

A new green competitive ELISA for aflatoxin M1 quantification in raw milk was developed. This diagnostic tool is based on an anti AFM1 mAb produced by plant molecular farming in alternative to classical systems. Our assay, showing an IC₅₀ below 25 ng/L, fits with the requirements of EU legislation limits for AFM1 (50 ng/L). Optimal accuracy was achieved in correspondence of the decision levels (25 and 50 ng/L), and the assay enabled AFM1 quantification in the range 5–110 ng/L, with limit of detection 3 ng/L. Moreover, to evaluate a real applicability in diagnostics, raw milk-spiked samples were analysed, achieving satisfactory recovery rates of AFM1. In conclusion, an efficient and ready-to-use diagnostic assay for the quantification of aflatoxin M1 in milk, based on a plant-produced recombinant mAb, has been successfully developed.

Summary: Immunodiagnostic systems for on‐site screening are increasingly in demand but require expensive reagents such as chemically modified antibodies. To obtain biomolecules as ready‐to‐use reagents for diagnostics, new recombinant fluorescent immunoglobulins G were generated by fusion of the heavy chain of a monoclonal antibody, 5H3, to two different fluorescent proteins: Green Fluorescent Protein (5H3GFP) and Cyan‐excitable Orange Fluorescent Protein (5H3CyOFP1). The engineered antibodies were expressed by transient agroinfiltration in Nicotiana benthamiana plants, used as bioreactor sustainable, cost‐effective and easy to scale up, with a yield of 80 and 35 mg/kg of purified 5H3GFP and 5H3CyOFP1, respectively. Both recombinant proteins retained the ability to recognize the antigen of the original mAb (aflatoxin M1, a contaminant of dairy products), while simultaneously being able to emit fluorescence in the green or orange light. To demonstrate their practical application in the diagnostic field, fluorescent antibodies were exploited to set up two fast screening systems for aflatoxin M1 detection: a lateral flow immunoassay and an OLED‐based device. For both diagnostic systems, antigen‐specific fluorescence signals as well as a first competitive assay were obtained. Although these assays will need a further implementation, our results demonstrate a concrete possibility to develop novel and fast analytical systems based on fluorescent recombinant antibodies produced in plants.