Optimised production of an anti-fungal antibody in Solanaceae hairy roots to develop new formulations against Candida albicans

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 Case of Advanced Tubal Ectopic Pregnancy after Emergency Contraception

Ectopic pregnancy is a relatively common condition and an important cause of morbidity in women of childbearing age. The most frequent implantation site is the fallopian tube. Most cases are diagnosed in an early gestational period. Patients come to the attention of clinicians for pelvic pain and vaginal blood loss, and consequent diagnosis is made through clinical presentation, laboratory tests, and ultrasound. Other rarer implantation sites such as the abdominal cavity give space for ectopic pregnancy to grow until later gestational ages, delaying diagnosis. This is a rare case of a healthy 41-year-old woman with an advanced ectopic pregnancy after emergency contraception with Ulipristal Acetate. The patient went to visit for amenorrhea after taking a contraceptive. Evaluation with ultrasound demonstrated a 10 + 4 weeks’ unruptured tubal pregnancy with fetal heart rate. The patient underwent laparoscopic salpingectomy without complication. This is the first case of such an advanced ectopic pregnancy in a woman who performed emergency contraception with Ulipristal Acetate

Production and Functional Characterization of a Recombinant Predicted Pore-Forming Protein (TVSAPLIP12) of Trichomonas vaginalis in Nicotiana benthamiana Plants

Pore-forming proteins (PFPs) are a group of functionally versatile molecules distributed in all domains of life, and several microbial pathogens notably use members of this class of proteins as cytotoxic effectors. Among pathogenic protists, Entamoeba histolytica, and Naegleria fowleri display a range of pore-forming toxins belonging to the Saposin-Like Proteins (Saplip) family: Amoebapores and Naegleriapores. Following the genome sequencing of Trichomonas vaginalis, we identified a gene family of 12 predicted saposin-like proteins (TvSaplips): this work focuses on investigating the potential role of TvSaplips as cytopathogenetic effectors. We provide evidence that TvSaplip12 gene expression is potently upregulated upon T. vaginalis contact with target cells. We cloned and expressed recombinant TvSaplip12 in planta and we demonstrate haemolytic, cytotoxic, and bactericidal activities of rTvSaplip12 in vitro. Also, evidence for TvSaplip subcellular discrete distribution in cytoplasmic granules is presented. Altogether, our results highlight the importance of TvSaplip in T. vaginalis pathogenesis, depicting its involvement in the cytolytic and bactericidal activities during the infection process, leading to predation on host cells and resident vaginal microbiota for essential nutrients acquisition. This hence suggests a potential key role for TvSaplip12 in T. vaginalis pathogenesis as a candidate Trichopore

Plant antibodies for human antifungal therapy

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

Contribution of the Microbial Communities Detected on an Oil Painting on Canvas to Its Biodeterioration

In this study, we investigated the microbial community (bacteria and fungi) colonising an oil painting on canvas, which showed visible signs of biodeterioration. A combined strategy, comprising culture-dependent and -independent techniques, was selected. The results derived from the two techniques were disparate. Most of the isolated bacterial strains belonged to related species of the phylum Firmicutes, as Bacillus sp. and Paenisporosarcina sp., whereas the majority of the non-cultivable members of the bacterial community were shown to be related to species of the phylum Proteobacteria, as Stenotrophomonas sp. Fungal communities also showed discrepancies: the isolated fungal strains belonged to different genera of the order Eurotiales, as Penicillium and Eurotium, and the non-cultivable belonged to species of the order Pleosporales and Saccharomycetales. The cultivable microorganisms, which exhibited enzymatic activities related to the deterioration processes, were selected to evaluate their biodeteriorative potential on canvas paintings; namely Arthrobacter sp. as the representative bacterium and Penicillium sp. as the representative fungus. With this aim, a sample taken from the painting studied in this work was examined to determine the stratigraphic sequence of its cross-section. From this information, “mock paintings,” simulating the structure of the original painting, were prepared, inoculated with the selected bacterial and fungal strains, and subsequently examined by micro-Fourier Transform Infrared spectroscopy, in order to determine their potential susceptibility to microbial degradation. The FTIR-spectra revealed that neither Arthrobacter sp. nor Penicillium sp. alone, were able to induce chemical changes on the various materials used to prepare “mock paintings.” Only when inoculated together, could a synergistic effect on the FTIR-spectra be observed, in the form of a variation in band position on the spectrum.The FTIR analyses performed in this study were financed by the Junta de Andalucía (RNM-325 group). The molecular analyses performed in this study were financed by the Austrian Science Fund (FWF) project ‘Hertha-Firnberg T137’ and the Spanish Ministry of Science and Innovation (Project CTQ2008-06727-C03-03). G. Piñar also thanks the “Elise-Richter V194-B20” projects